Nanoparticle comprising rapamycin and albumin as anticancer agent

ABSTRACT

The present invention features methods for treating, stabilizing, preventing, and/or delaying cancer by administering nanoparticles that comprise rapamycin or a derivative thereof. The invention also provides compositions (e.g., unit dosage forms) comprising nanoparticles that comprise a carrier protein and rapamycin or a derivative thereof. The invention further provides combination therapy methods of treating cancer comprising administering to an individual an effective amount of nanoparticles that comprise rapamycin or a derivative thereof and a second therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit to provisional application Ser.No. 60/905,669, filed Mar. 7, 2007, provisional application Ser. No.60/905,734, filed Mar. 7, 2007, provisional application Ser. No.60/905,662, filed Mar. 7, 2007, provisional application Ser. No.60/905,735, filed Mar. 7, 2007, provisional application Ser. No.60/905,672, filed Mar. 7, 2007, provisional application Ser. No.60/905,787, filed Mar. 7, 2007, provisional application Ser. No.60/905,663, filed Mar. 7, 2007, provisional application Ser. No.60/905,767, filed Mar. 7, 2007, provisional application Ser. No.60/905,750, filed Mar. 7, 2007, provisional application Ser. No.60/923,248, filed Apr. 13, 2007, and provisional application Ser. No.60/923,456, filed Apr. 13, 2007, the entire disclosures of each of whichare hereby incorporated by reference.

TECHNICAL FIELD

This application relates to methods and compositions for treating,stabilizing, preventing, and/or delaying cancer using nanoparticles thatcomprise rapamycin or a derivative thereof. The application furtherprovides combination therapy methods of treating cancer comprisingadministering to an individual an effective amount of nanoparticles thatcomprise rapamycin or a derivative thereof and a second therapy.

BACKGROUND

The failure of a significant number of tumors to respond to drug and/orradiation therapy is a serious problem in the treatment of cancer. Infact, this is one of the main reasons why many of the most prevalentforms of human cancer still resist effective chemotherapeuticintervention, despite certain advances in the field of chemotherapy.

Cancer is now primarily treated with one or a combination of three typesof therapies: surgery, radiation, and chemotherapy. Surgery is atraditional approach in which all or part of a tumor is removed from thebody. Surgery generally is only effective for treating the earlierstages of cancer. While surgery is sometimes effective in removingtumors located at certain sites, for example, in the breast, colon, andskin, it cannot be used in the treatment of tumors located in otherareas, inaccessible to surgeons, nor in the treatment of disseminatedneoplastic conditions such as leukemia. For more than 50% of cancerindividuals, by the time they are diagnosed they are no longercandidates for effective surgical treatment. Surgical procedures mayincrease tumor metastases through blood circulation during surgery. Mostof cancer individuals do not die from the cancer at the time ofdiagnosis or surgery, but rather die from the metastasis and therecurrence of the cancer.

Other therapies are also often ineffective. Radiation therapy is onlyeffective for individuals who present with clinically localized diseaseat early and middle stages of cancer, and is not effective for the latestages of cancer with metastasis. Radiation is generally applied to adefined area of the subject's body which contains abnormal proliferativetissue, in order to maximize the dose absorbed by the abnormal tissueand minimize the dose absorbed by the nearby normal tissue. However, itis difficult (if not impossible) to selectively administer therapeuticradiation to the abnormal tissue. Thus, normal tissue proximate to theabnormal tissue is also exposed to potentially damaging doses ofradiation throughout the course of treatment. The efficacy ofradiotherapeutic techniques in destroying abnormal proliferative cellsis therefore balanced by associated cytotoxic effects on nearby normalcells. Because of this, radiotherapy techniques have an inherentlynarrow therapeutic index which results in the inadequate treatment ofmost tumors. Even the best radiotherapeutic techniques may result inincomplete tumor reduction, tumor recurrence, increased tumor burden,and induction of radiation resistant tumors.

Chemotherapy involves the disruption of cell replication or cellmetabolism. Chemotherapy can be effective, but there are severe sideeffects, e.g., vomiting, low white blood cells, loss of hair, loss ofweight and other toxic effects. Because of the extremely toxic sideeffects, many cancer individuals cannot successfully finish a completechemotherapy regime. Chemotherapy-induced side effects significantlyimpact the quality of life of the individual and may dramaticallyinfluence individual compliance with treatment. Additionally, adverseside effects associated with chemotherapeutic agents are generally themajor dose-limiting toxicity in the administration of these drugs. Forexample, mucositis is one of the major dose limiting toxicities forseveral anticancer agents, including 5-FU, methotrexate, and antitumorantibiotics, such as doxorubicin. Many of these chemotherapy-inducedside effects if severe may lead to hospitalization, or require treatmentwith analgesics for the treatment of pain. Some cancer individuals diefrom the chemotherapy due to poor tolerance to the chemotherapy. Theextreme side effects of anticancer drugs are caused by the poor targetspecificity of such drugs. The drugs circulate through most normalorgans of individuals as well as intended target tumors. The poor targetspecificity that causes side effects also decreases the efficacy ofchemotherapy because only a fraction of the drugs is correctly targeted.The efficacy of chemotherapy is further decreased by poor retention ofthe anti-cancer drugs within the target tumors.

Another problem associated with chemotherapy is the development of drugresistance. Drug resistance is the name given to the circumstances whena disease does not respond to a treatment drug or drugs. Drug resistancecan be either intrinsic, which means that disease has never beenresponsive to the drug or drugs, or it can be acquired, which means thedisease ceases responding to a drug or drugs that the disease hadpreviously been responsive. Combination therapy, including combinationchemotherapy, has the potential advantages of both avoiding theemergence of resistant cells and to kill pre-existing cells which arealready drug resistant.

Due to the limitations of current treatments for cancer, the severityand breadth of neoplasm, tumor and cancer, there remains a significantinterest in and need for additional or alternative therapies fortreating, stabilizing, preventing, and/or delaying cancer. Preferably,the treatments overcome the shortcomings of current surgical,chemotherapy, and radiation treatments.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for the treatment of cancer usingnanoparticles that comprise rapamycin or a derivative thereof.Accordingly, the invention in some embodiments provides a method oftreating cancer in an individual by administering to the individual(e.g., a human) an effective amount of a composition comprisingnanoparticles that comprise rapamycin or a derivative thereof and acarrier protein. In some embodiments, the cancer is early stage cancer,non-metastatic cancer, primary cancer, advanced cancer, locally advancedcancer, metastatic cancer, cancer in remission, recurrent cancer, cancerin an adjuvant setting, cancer in a neoadjuvant setting, or cancersubstantially refractory to hormone therapy. In some embodiments, thecancer is a solid tumor. In some embodiments, the cancer is not a solidtumor (i.e., other than a solid tumor). In some embodiments, the canceris a plasmacytoma. In some embodiments, the cancer is multiple myeloma,renal cell carcinoma, prostate cancer, lung cancer, melanoma, braincancer (e.g., glioblastoma), ovarian cancer, or breast cancer. In someembodiments, the cancer is not a carcinoma (i.e., other than acarcinoma). In some embodiments, the cancer is not colon cancer (i.e.,other than colon cancer). In some embodiments, the cancer is not breastcancer (i.e., other than breast cancer). In some embodiments, the canceris not ovarian cancer, prostate cancer, or brain cancer. In someembodiments, one or more symptoms of the cancer are ameliorated. In someembodiments, the cancer is delayed or prevented.

In some embodiments, the amount of the rapamycin or derivative thereofin the effective amount of the composition is in the range of about 54mg to about 540 mg, such as about 180 mg to about 270 mg or about 216mg. In some embodiments, the rapamycin or derivative thereof isadministered parenterally (e.g., intravenously). In some embodiments, ataxane is not administered to the individual (i.e., other than ataxane). In some embodiments, the taxane administered is not ananoparticle taxane composition. In some embodiments, the rapamycin orderivative thereof is the only pharmaceutically active agent for thetreatment of cancer that is administered to the individual. In someembodiments, rapamycin is administered. In some embodiments, thecomposition comprises more than about 50% of the rapamycin or derivativethereof in nanoparticle form. In some embodiments, the carrier proteinis albumin, such as human serum albumin. In some embodiments, theaverage diameter of the nanoparticles in the composition is no greaterthan about 200 nm (such as no greater than about 100 nm). In someembodiments, the nanoparticle compositions are sterile filterable. Insome embodiments, the weight ratio of the carrier protein to therapamycin or derivative thereof in the nanoparticles is less than about18:1. In some embodiments, the weight ratio of the carrier protein tothe rapamycin or derivative thereof in the nanoparticle compositions isless than about 18:1.

The invention also provides pharmaceutical compositions such as unitdosage forms that are useful for treating cancer. Accordingly, theinvention in some embodiments provides a pharmaceutical composition(e.g., a unit dosage form of a pharmaceutical composition) that includesnanoparticles that comprise rapamycin or a derivative thereof and acarrier protein. In some embodiments, the composition also includes apharmaceutically acceptable carrier. In various embodiments, the canceris early stage cancer, non-metastatic cancer, primary cancer, advancedcancer, locally advanced cancer, metastatic cancer, cancer in remission,recurrent cancer, cancer in an adjuvant setting, cancer in a neoadjuvantsetting, or cancer substantially refractory to hormone therapy. In someembodiments, the cancer is a solid tumor. In some embodiments, thecancer is not a solid tumor (i.e., other than a solid tumor). In someembodiments, the cancer is a plasmacytoma. In some embodiments, thecancer is multiple myeloma, renal cell carcinoma, prostate cancer, lungcancer, melanoma, brain cancer (e.g., glioblastoma), ovarian cancer, orbreast cancer. In some embodiments, the cancer is not a carcinoma (i.e.,other than a carcinoma). In some embodiments, the cancer is not coloncancer (i.e., other than colon cancer). In some embodiments, the canceris not breast cancer (i.e., other than breast cancer). In someembodiments, the cancer is not ovarian cancer, prostate cancer, or braincancer. In some embodiments, one or more symptoms of the cancer areameliorated. In some embodiments, the cancer is delayed or prevented.

In some embodiments, the amount of the rapamycin or derivatives thereofin the composition (e.g., a dose or a unit dosage form) is in the rangeof about 54 mg to about 540 mg, such as about 180 mg to about 270 mg, orabout 216 mg. In some embodiments, the carrier is suitable forparenteral administration (e.g., intravenous administration). In someembodiments, a taxane is not contained in the composition. In someembodiments, the rapamycin or derivative thereof is the onlypharmaceutically active agent for the treatment of cancer that iscontained in the composition. In some embodiments, the compositioncomprises rapamycin. In some embodiments, the composition comprises morethan about 50% of the rapamycin or derivative thereof in nanoparticleform. In some embodiments, the carrier protein is albumin, such as humanserum albumin. In some embodiments, the average diameter of thenanoparticles in the composition is no greater than about 200 nm (suchas no greater than about 100 nm). In some embodiments, the nanoparticlecompositions are sterile filterable. In some embodiments, the weightratio of the carrier protein to the rapamycin or derivative thereof inthe nanoparticles is less than about 18:1. In some embodiments, theweight ratio of the carrier protein to the rapamycin or derivativethereof in the nanoparticle compositions is less than about 18:1.

In yet another aspect, the invention includes a kit with (i) acomposition comprising nanoparticles that comprise rapamycin or aderivative thereof and a carrier protein and (ii) instructions for usein treating cancer. In various embodiments, the cancer is early stagecancer, non-metastatic cancer, primary cancer, advanced cancer, locallyadvanced cancer, metastatic cancer, cancer in remission, recurrentcancer, cancer in an adjuvant setting, cancer in a neoadjuvant setting,or cancer substantially refractory to hormone therapy. In someembodiments, the cancer is a solid tumor. In some embodiments, thecancer is a plasmacytoma. In some embodiments, the cancer is multiplemyeloma, renal cell carcinoma, prostate cancer, lung cancer, melanoma,brain cancer (e.g. glioblastoma), ovarian cancer, or breast cancer. Insome embodiments, the cancer is not colon cancer. In some embodiments,the cancer is not breast cancer. In some embodiments, one or moresymptoms of the cancer are ameliorated. In some embodiments, the canceris delayed or prevented.

In some embodiments, the amount of the rapamycin or derivative thereofin the kit is in the range of about 54 mg to about 540 mg, such as about180 mg to about 270 mg or about 216 mg. In some embodiments, therapamycin or derivative thereof is administered parenterally (e.g.,intravenously). In some embodiments, the kit does not contain a taxane.In some embodiments, the rapamycin or derivative thereof is the onlypharmaceutically active agent for the treatment of cancer that iscontained in the kit. In some embodiments, the kit comprises anotherpharmaceutically active agent for the treatment of cancer. In someembodiments, the other pharmaceutically active agent is achemotherapeutic agent. In some embodiments, the kit comprisesrapamycin. In some embodiments, the composition comprises more thanabout 50% of the rapamycin or derivative thereof in nanoparticle form.In some embodiments, the carrier protein is albumin, such as human serumalbumin. In some embodiments, the average diameter of the nanoparticlesin the composition is no greater than about 200 nm (such as no greaterthan about 100 nm). In some embodiments, the nanoparticle compositionsare sterile filterable. In some embodiments, the weight ratio of thecarrier protein to the rapamycin or derivative thereof in thenanoparticles is less than about 18:1. In some embodiments, the weightratio of the carrier protein to the rapamycin or derivative thereof inthe nanoparticle compositions is less than about 18:1.

The present invention also provides methods for the treatment of cancerusing combination therapies. The invention provides a method of treatingcancer comprising a) a first therapy comprising administering to anindividual an effective amount of a composition comprising nanoparticlescomprising rapamycin or a derivative thereof and a carrier protein andb) a second therapy, such as surgery, radiation, gene therapy,immunotherapy, bone marrow transplantation, stem cell transplantation,hormone therapy, targeted therapy, cryotherapy, ultrasound therapy,photodynamic therapy, and/or chemotherapy (e.g., one or more compoundsor pharmaceutically acceptable salts thereof useful for treatingcancer).

In some embodiments, the invention provides a method of treating cancerin an individual comprising administering to the individual a) aneffective amount of a composition comprising nanoparticles comprising arapamycin or a derivative thereof and a carrier protein and b) aneffective amount of at least one other chemotherapeutic agent. In someembodiments, the chemotherapeutic agent is any of (and in someembodiments selected from the group consisting of) taxane,antimetabolites (including nucleoside analogs), platinum-based agents,alkylating agents, tyrosine kinase inhibitors, anthracyclineantibiotics, vinca alkloids, proteasome inhibitors, modulators ofHER2/neu, modulators of EGFR, modulators of VEGFR, and topoisomeraseinhibitors. In some embodiments, the chemotherapeutic agent is aplatinum-based agent, such as carboplatin. In some embodiments, thechemotherapeutic agent is a modulator of HER2/neu (such as an inhibitorof HER2/neu for example Herceptin®). In some embodiments, thechemotherapeutic agent is a modulator of EGFR (such as an inhibitor ofEGFR for example Erbitux®). In some embodiments, the chemotherapeuticagent is an anti-VEGF antibody (such as bevacizumab, e.g., Avastin®). Insome embodiments, the effective amounts of the nanoparticle compositionand the anti-VEGF antibody synergistically inhibit cell proliferation ormetastasis. In some embodiments, the chemotherapeutic agent affects asignaling pathway involving a target of rapamycin. In some embodimentsthe chemotherapeutic agent affects a signaling pathway involving mTOR(such as the PI3K/Akt signaling pathway). In some embodiments, a taxaneis not administered to the individual. In some embodiments, the taxaneadministered is not in a nanoparticle composition.

In some embodiments, the composition comprising nanoparticles comprisinga rapamycin or a derivative thereof and a carrier protein and thechemotherapeutic agent are administered simultaneously, either in thesame composition or in separate compositions. In some embodiments, thenanoparticle composition comprising a rapamycin or a derivative thereofand a carrier protein and the chemotherapeutic agent are administeredsequentially, e.g., the nanoparticle composition is administered eitherprior to or after the administration of the chemotherapeutic agent. Insome embodiments, the administration'of the nanoparticle compositioncomprising a rapamycin or a derivative thereof and a carrier protein andthe chemotherapeutic agent are concurrent, e.g., the administrationperiod of the nanoparticle composition and that of the chemotherapeuticagent overlap with each other. In some embodiments, the administrationof the nanoparticle composition comprising a rapamycin or a derivativethereof and a carrier protein and the chemotherapeutic agent arenon-concurrent. For example, in some embodiments, the administration ofthe nanoparticle composition comprising a rapamycin or a derivativethereof and a carrier protein is terminated before the chemotherapeuticagent is administered. In some embodiments, the administration of thechemotherapeutic agent is terminated before the nanoparticle compositioncomprising a rapamycin or a derivative thereof and a carrier protein isadministered.

In some embodiments, there is provided a method of treating cancer in anindividual comprising a) a first therapy comprising administering to theindividual a composition comprising nanoparticles comprising rapamycinor a derivative thereof and a carrier protein, and b) a second therapycomprising surgery, radiation, gene therapy, immunotherapy, bone marrowtransplantation, stem cell transplantation, hormone therapy, targetedtherapy, cryotherapy, ultrasound therapy, photodynamic therapy, orcombinations thereof. In some embodiments, the second therapy is hormonetherapy. In some embodiments, the second therapy is radiation therapy.In some embodiments, the second therapy is surgery. In some embodiments,the first therapy is carried out prior to the second therapy. In someembodiments, the first therapy is carried out after the second therapy.

In some embodiments, the cancer being treated by combination therapy isearly stage cancer, non-metastatic cancer, primary cancer, advancedcancer, locally advanced cancer, metastatic cancer, cancer in remission,recurrent cancer, cancer in an adjuvant setting, cancer in a neoadjuvantsetting, or cancer substantially refractory to hormone therapy. In someembodiments, the cancer is a solid tumor. In some embodiments, thecancer is not a solid tumor (i.e., other than a solid tumor). In someembodiments, the cancer is a plasmacytoma. In some embodiments, thecancer is multiple myeloma, renal cell carcinoma, prostate cancer, lungcancer, melanoma, brain cancer (e.g., glioblastoma), ovarian cancer, orbreast cancer. In some embodiments, the cancer is not a carcinoma (i.e.,other than a carcinoma). In some embodiments, the cancer is not coloncancer (i.e., other than colon cancer). In some embodiments, the canceris not breast cancer (i.e., other than breast cancer). In someembodiments, the cancer is not ovarian cancer, prostate cancer, or braincancer. In some embodiments, one or more symptoms of the cancer areameliorated. In some embodiments, the cancer is delayed or prevented.

In some embodiments, the amount of the rapamycin or derivative thereofin the effective amount of the composition used in combination therapyis in the range of about 54 mg to about 540 mg, such as about 180 mg toabout 270 mg or about 216 mg. In some embodiments, the rapamycin orderivative thereof is administered parenterally (e.g., intravenously).In some embodiments, a taxane is not administered to the individual(i.e., other than taxane). In some embodiments, the taxane administeredis not a nanoparticle taxane composition. In some embodiments, rapamycinis administered. In some embodiments, the composition comprises morethan about 50% of the rapamycin or derivative thereof in nanoparticleform. In some embodiments, the carrier protein is albumin, such as humanserum albumin. In some embodiments, the average diameter of thenanoparticles in the composition is no greater than about 200 nm (suchas no greater than about 100 nm). In some embodiments, the nanoparticlecompositions are sterile filterable. In some embodiments, the weightratio of the carrier protein to the rapamycin or derivative thereof inthe nanoparticles is less than about 18:1. In some embodiments, theweight ratio of the carrier protein to the rapamycin or derivativethereof in the nanoparticle compositions is less than about 18:1.

The invention also provides pharmaceutical compositions such as unitdosage forms that are useful in combination therapy for treating cancer.Accordingly, the invention in some embodiments provides a pharmaceuticalcomposition (e.g., a unit dosage form of a pharmaceutical composition)for use in combination therapy that includes nanoparticles that compriserapamycin or a derivative thereof and a carrier protein. In someembodiments, the pharmaceutical composition includes a) nanoparticlescomprising rapamycin or a derivative thereof and a carrier protein andb) at least one other therapeutic agent. In some embodiments, the othertherapeutic agent comprises a chemotherapeutic agent. In someembodiments, the other therapeutic agent comprises a hormone therapeuticagent. In some embodiments, the composition also includes apharmaceutically acceptable carrier. In some embodiments, the cancer isearly stage cancer, non-metastatic cancer, primary cancer, advancedcancer, locally advanced cancer, metastatic cancer, cancer in remission,recurrent cancer, cancer in an adjuvant setting, cancer in a neoadjuvantsetting, or cancer substantially refractory to hormone therapy. In someembodiments, the cancer is a solid tumor. In some embodiments, thecancer is not a solid tumor (i.e., other than a solid tumor). In someembodiments, the cancer is a plasmacytoma. In some embodiments, thecancer is multiple myeloma, renal cell carcinoma, prostate cancer, lungcancer, melanoma, brain cancer (e.g., glioblastoma), ovarian cancer, orbreast cancer. In some embodiments, the cancer is a carcinoma (i.e.,other than a carcinoma). In some embodiments, the cancer is not coloncancer (i.e., other than colon cancer). In some embodiments, the canceris not breast cancer (i.e., other than breast cancer). In someembodiments, the cancer is not ovarian cancer, prostate cancer, or braincancer. In some embodiments, one or more symptoms of the cancer areameliorated. In some embodiments, the cancer is delayed or prevented.

In some embodiments, the amount of the rapamycin or derivatives thereofin the composition (e.g., a dose or a unit dosage form) for use incombination therapy is in the range of about 54 mg to about 540 mg, suchas about 180 mg to about 270 mg, or about 216 mg. In some embodiments,the carrier is suitable for parenteral administration (e.g., intravenousadministration). In some embodiments, a taxane is not contained in thecomposition. In some embodiments, the rapamycin or derivative thereof isthe only pharmaceutically active agent for the treatment of cancer thatis contained in the composition (for example, as part of a kit thatcontains instructions for using the composition with another therapy).

In some embodiments, the composition comprises rapamycin. In someembodiments, the composition comprises more than about 50% of therapamycin or derivative thereof in nanoparticle form. In someembodiments, the carrier protein is albumin, such as huam serum albumin.In some embodiments, the average diameter of the nanoparticles in thecomposition is no greater than about 200 nm (such as no greater thanabout 100 nm). In some embodiments, the nanoparticle compositions aresterile filterable. In some embodiments, the weight ratio of the carrierprotein to the rapamycin or derivative thereof in the nanoparticles isless than about 18:1. In some embodiments, the weight ratio of thecarrier protein to the rapamycin or derivative thereof in thenanoparticle compositions is less than about 18:1.

In yet another aspect, the invention includes a kit with (i) acomposition comprising nanoparticles that comprise rapamycin or aderivative thereof and a carrier protein and (ii) instructions for usein combination therapy for treating cancer. The invention also provideskits for using the rapamycin (or its derivatives) compositions describedherein in combination therapy context. For example, a kit may providesuch a composition in addition to another therapeutic composition. Insome embodiments, the instructions are instructions for providing afirst and second therapy, wherein either the first or second therapycomprises administering a composition that comprises nanoparticles ofrapamycin or derivative thereof and a carrier protein. In someembodiments, the kit further comprises at least one other therapeuticagent. In some embodiments, the other therapeutic agent comprises achemotherapeutic agent. In some embodiments, the other therapeutic agentcomprises a hormone therapeutic agent. In some embodiments, the canceris early stage cancer, non-metastatic cancer, primary cancer, advancedcancer, locally advanced cancer, metastatic cancer, cancer in remission,recurrent cancer, cancer in an adjuvant setting, cancer in a neoadjuvantsetting, or cancer substantially refractory to hormone therapy. In someembodiments, the cancer is a solid tumor. In some embodiments, thecancer is not a solid tumor (i.e., other than a solid tumor). In someembodiments, the cancer is a plasmacytoma. In some embodiments, thecancer is multiple myeloma, renal cell carcinoma, prostate cancer, lungcancer, melanoma, brain cancer (e.g., glioblastoma), ovarian cancer, orbreast cancer. In some embodiments, the cancer is a carcinoma (i.e.,other than a carcinoma). In some embodiments, the cancer is not coloncancer (i.e., other than colon cancer). In some embodiments, the canceris not breast cancer (i.e., other than breast cancer). In someembodiments, the cancer is not ovarian cancer, prostate cancer, or braincancer. In some embodiments, one or more symptoms of the cancer areameliorated. In some embodiments, the cancer is delayed or prevented.

In some embodiments, the amount of the rapamycin or derivative thereofin the kit for use in combination therapy is in the range of about 54 mgto about 540 mg, such as about 180 mg to about 270 mg or about 216 mg.In some embodiments, the rapamycin or derivative thereof is administeredparenterally (e.g., intravenously). In some embodiments, the kit doesnot contain a taxane. In some embodiments, the rapamycin or derivativethereof is the only pharmaceutically active agent for the treatment ofcancer that is contained in the kit. In some embodiments, the kitcomprises another pharmaceutically active agent for the treatment ofcancer. In some embodiments, the other pharmaceutically active agent isa chemotherapeutic agent. In some embodiments, the kit comprisesrapamycin. In some embodiments, the composition comprises more thanabout 50% of the rapamycin or derivative thereof in nanoparticle form.In some embodiments, the carrier protein is albumin, such as human serumalbumin. In some embodiments, the average diameter of the nanoparticlesin the composition is no greater than about 200 nm (such as no greaterthan about 100 nm). In some embodiments, the nanoparticle compositionsare sterile filterable. In some embodiments, the weight ratio of thecarrier protein to the rapamycin or derivative thereof in thenanoparticles is less than about 18:1. In some embodiments, the weightratio of the carrier protein to the rapamycin or derivative thereof inthe nanoparticle compositions is less than about 18:1.

The invention also provides any of the compositions described (e.g., acomposition comprising nanoparticles that comprise rapamycin or aderivative thereof and a carrier protein) for any use described hereinwhether in the context of use as a medicament and/or use for manufactureof a medicament. Also provided are unit dosage forms of compositionsdescribed herein, articles of manufacture comprising the inventivecompositions or unit dosage forms in suitable packaging (e.g., vials orvessels including sealed vials or vessels and sterile sealed vials orvessels), and kits comprising the unit dosage forms. The invention alsoprovides methods of making and using these compositions as describedherein.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the present invention.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a table listing the intravenous pharmacokinetic parameters forthe albumin-containing nanoparticle formulation of rapamycin(hereinafter referred to as Nab-rapamycin).

FIG. 2A is a graph of Cmaxversus dose, showing linearity for theNab-rapamycin.

FIG. 2B is a graph of AUC versus dose, showing linearity forNab-rapamycin.

FIG. 2C is a graph of Vss versus dose, showing possible saturable volumeof distribution for Nab-rapamycin.

FIG. 2D is a graph showing the log-linear plot of Nab-rapamycin bloodconcentration vs. time following IV administration to rats at doselevels of 15 mg/kg, 30 mg/kg, and 45 mg/kg.

FIG. 3A is a graph of the antitumor activity of Nab-rapamycin in micewith MX-1 breast tumor xenografts.

FIG. 3B is a graph of the weight loss in mice with MX-1 breast tumorxenografts after the administration of Nab-rapamycin or saline.

FIG. 4 is a graph showing the antitumor activity of Abraxane™,Nab-rapamycin, and Nab-rapamycin in combination with Abraxane™ in micewith HT29 colon tumor xenografts.

FIG. 5A is a graph showing the antitumor activity of Nab-rapamycin inmice with HT29 colon tumor xenografts.

FIG. 5B is a graph showing the weight loss in mice with H29 colon tumorxenografts after the administration of Nab-rapamycin or DMSO.

FIG. 6A is a graph showing the antitumor activity of Nab-rapamycin inmice with HCT-116 colon tumor xenografts.

FIG. 6B is a graph showing the weight loss in mice with HCT-116 colontumor xenografts after the administration of Nab-rapamycin or saline.

FIG. 7 is a graph showing the antitumor activity of Nab-rapamycin inmice with MM1S multiple myeloma tumor xenografts.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods, compositions, and kits for thetreatment or prevention of cancer using nanoparticles that compriserapamycin or a derivative thereof and a carrier protein (such asalbumin). The present invention further provides methods, compositions,and kits for use in combination therapy for the treatment or preventionof cancer using nanoparticles that comprise rapamycin or a derivativethereof and a carrier protein (such as albumin). Any of thesecompositions can be used to treat, stabilize, prevent, and/or delaycancer.

In particular, nanoparticles comprising rapamycin (also referred to as a“nanoparticle composition”) and the carrier protein albumin were shownto significantly inhibit the growth of a human mammary carcinomaimplanted into a mouse model (Example 3) and inhibit tumor growth inmice with MM1S multiple myeloma tumor xenografts (Example 12B). Thisalbumin-containing nanoparticle formulation of rapamycin was nontoxic atthe doses tested and displayed linear pharmacokinetics with respect todose (Example 2). The nanoparticle formulation of albumin and rapamycinenhances tumor penetration through albumin receptor (gp60)-mediatedbinding of the SPARC protein, which is upregulated in some cancer cells(e.g., breast cancer cells). This increased specificity of Nab-rapamycinmay increase the effectiveness of rapamycin and may allow lower doses ofrapamycin to be used, which would minimize toxic effects from rapamycinwhile still inhibiting, stabilizing, preventing, or delaying tumorgrowth. The increased specificity may also reduce toxic side-effectsfrom interactions of rapamycin with noncancerous cells and tissues, suchas intestinal toxicity that sometimes limits the dose of rapamycin thatcan be given to a patient. The nanoparticle formulation of rapamycinalso increases the solubility of rapamycin and allows larger doses to beused, if desired.

Definitions

As used herein, “the composition” or “compositions” includes and isapplicable to compositions of the invention. The invention also providespharmaceutical compositions comprising the components described herein.

The term, “rapamycin” herein refers to rapamycin or its derivatives andaccordingly the invention contemplates and includes all theseembodiments. Rapamycin is sometimes referred to elsewhere as sirolimus,rapammune, or rapamune. Reference to “rapamycin” is to simplify thedescription and is exemplary. Derivatives of rapamycin include, but arenot limited to, compounds that are structurally similar to rapamycin, orare in the same general chemical class as rapamycin, analogs ofrapamycin, or pharmaceutically acceptable salts of rapamycin or itsderivatives or analogs. In some embodiments, rapamycin or a derivativethereof increases basal AKT activity, increases AKT phosphorylation,increases PI3-kinase activity, increases the length of activation of AKT(e.g., activation induced by exogenous IGF-1), inhibits serinephosphorylation of IRS-1, inhibits IRS-1 degradation, inhibits or altersCXCR4 subcellular localization, inhibits VEGF secretion, decreasesexpression of cyclin D2, decreases expression of survivin, inhibitsIL-6-induced multiple myeloma cell growth, inhibits cancer cellproliferation, increases apoptosis, increases cell cycle arrest,increases cleavage of poly(ADPribose) polymerase, increases cleavage ofcaspase-8/caspase-9, alters or inhibits signaling in thephosphatidylinositol 3-kinase/AKT/mTOR and/or cyclin D1/retinoblastomapathways, inhibits angiogenesis, and/or inhibits osteoclast formation.In some embodiments, the derivative of rapamycin retains one or moresimilar biological, pharmacological, chemical and/or physical properties(including, for example, functionality) as rapamycin. In someembodiments, the rapamycin derivative has at least about any of 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of an activity ofrapamycin. For example, the decrease in the size of a tumor, the numberof cancer cells, or the growth rate of a tumor caused by a rapamycinderivative is preferably at least about any of 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% or 100% of the corresponding decrease caused bythe same amount of rapamycin. An exemplary rapamycin derivative includesbenzoyl rapamycin, such as that disclosed in paragraph [0022] of WO2006/089207, which is hereby incorporated by reference in its entirety.Other exemplary rapamycin derivatives include WY-090217, AY-22989,NSC-226080, SiiA-9268A, oxaazacyclohentriacontine, temsirolimus (CCI 779(Wyeth)), everolimus (RAD 001 (Novartis)), pimecrolimus (ASM981),SDZ-RAD, SAR943, ABT-578, AP23573, and Biolimus A9.

Unless clearly indicated otherwise, “an individual” as used hereinintends a mammal, including but not limited to a primate, human, bovine,horse, feline, canine, or rodent.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results including clinical results. For purposesof this invention, beneficial or desired clinical results include, butare not limited to, one or more of the following: decreasing one moresymptoms resulting from the disease, diminishing the extent of thedisease, stabilizing the disease (e.g., preventing or delaying theworsening of the disease), preventing or delaying the spread (e.g.,metastasis) of the disease, preventing or delaying the occurrence orrecurrence of the disease, delay or slowing the progression of thedisease, ameliorating the disease state, providing a remission (whetherpartial or total) of the disease, decreasing the dose of one or moreother medications required to treat the disease, delaying theprogression of the disease, increasing the quality of life, and/orprolonging survival. In some embodiments, the composition reduces theseverity of one or more symptoms associated with cancer by at leastabout any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%compared to the corresponding symptom in the same subject prior totreatment or compared to the corresponding symptom in other subjects notreceiving the composition. Also encompassed by “treatment” is areduction of pathological consequence of cancer. The methods of theinvention contemplate any one or more of these aspects of treatment.

As used herein, “delaying” the development of cancer means to defer,hinder, slow, retard, stabilize, and/or postpone development of thedisease. This delay can be of varying lengths of time, depending on thehistory of the disease and/or individual being treated. As is evident toone skilled in the art, a sufficient or significant delay can, ineffect, encompass prevention, in that the individual does not developthe disease. A method that “delays” development of cancer is a methodthat reduces probability of disease development in a given time frameand/or reduces the extent of the disease in a given time frame, whencompared to not using the method. Such comparisons are typically basedon clinical studies, using a statistically significant number ofsubjects. Cancer development can be detectable using standard methods,such as routine physical exams, mammography, imaging, or biopsy.Development may also refer to disease progression that may be initiallyundetectable and includes occurrence, recurrence, and onset.

As used herein, an “at risk” individual is an individual who is at riskof developing cancer. An individual “at risk” may or may not havedetectable disease, and may or may not have displayed detectable diseaseprior to the treatment methods described herein. “At risk” denotes thatan individual has one or more so-called risk factors, which aremeasurable parameters that correlate with development of cancer, whichare described herein. An individual having one or more of these riskfactors has a higher probability of developing cancer than an individualwithout these risk factor(s).

“Adjuvant setting” refers to a clinical setting in which an individualhas had a history of cancer, and generally (but not necessarily) beenresponsive to therapy, which includes, but is not limited to, surgery(e.g., surgical resection), radiotherapy, and chemotherapy. However,because of their history of the cancer, these individuals are consideredat risk of development of the disease. Treatment or administration inthe “adjuvant setting” refers to a subsequent mode of treatment. Thedegree of risk (e.g., when an individual in the adjuvant setting isconsidered as “high risk” or “low risk”) depends upon several factors,most usually the extent of disease when first treated.

“Neoadjuvant setting” refers to a clinical setting in which the methodis be carried out before the primary/definitive therapy.

As used herein, by “pharmaceutically active compound” is meant achemical compound that induces a desired effect, e.g., treating,stabilizing, preventing, and/or delaying cancer.

As used herein, by “combination therapy” is meant a first therapy thatincludes nanoparticles comprising rapamycin or a derivative thereof anda carrier protein in conjunction with a second therapy (e.g., radiation,surgery, or chemotherapeutic agent) useful for treating, stabilizing,preventing, and/or delaying cancer. Administration in “conjunction with”another compound includes administration in the same or differentcomposition(s), either sequentially, simultaneously, or continuously. Insome variations, the combination therapy optionally includes one or morepharmaceutically acceptable carriers or excipients, non-pharmaceuticallyactive compounds, and/or inert substances.

The term “effective amount” intends such amount of a composition (e.g.,nanoparticles that comprise rapamycin or a derivative thereof and acarrier protein), first therapy, second therapy, or a combinationtherapy, which in combination with its parameters of efficacy andtoxicity, should be effective in a given therapeutic form based on theknowledge of the practicing specialist. In various embodiments, aneffective amount of the composition or therapy may (i) reduce the numberof cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow tosome extent, and preferably stop cancer cell infiltration intoperipheral organs; (iv) inhibit (e.g., slow to some extent andpreferably stop) tumor metastasis; (v) inhibit tumor growth; (vi)prevent or delay occurrence and/or recurrence of a tumor; and/or (vii)relieve to some extent one or more of the symptoms associated with thecancer. In various embodiments, the amount is sufficient to ameliorate,palliate, lessen, and/or delay one or more of symptoms of cancer.

In some embodiments, the amount of the composition, first therapy,second therapy, or combination therapy is an amount sufficient todecrease the size of a tumor, decrease the number of cancer cells, ordecrease the growth rate of a tumor by at least about any of 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to thecorresponding tumor size, number of cancer cells, or tumor growth ratein the same subject prior to treatment or compared to the correspondingactivity in other subjects not receiving the treatment. Standard methodscan be used to measure the magnitude of this effect, such as in vitroassays with purified enzyme, cell-based assays, animal models, or humantesting.

As is understood in the art, an “effective amount” may be in one or moredoses, i.e., a single dose or multiple doses may be required to achievethe desired treatment endpoint. An effective amount may be considered inthe context of administering one or more therapeutic agents, and ananoparticle composition (e.g., a composition including rapamycin and acarrier protein) may be considered to be given in an effective amountif, in conjunction with one or more other agents, a desirable orbeneficial result may be or is achieved. The components (e.g., the firstand second therapies) in a combination therapy of the invention may beadministered sequentially, simultaneously, or continuously using thesame or different routes of administration for each component. Thus, aneffective amount of a combination therapy includes an amount of thefirst therapy and an amount of the second therapy that when administeredsequentially, simultaneously, or continuously produces a desiredoutcome.

A “therapeutically effective amount” refers to an amount of acomposition (e.g., nanoparticles that comprise rapamycin or a derivativethereof and a carrier protein), first therapy, second therapy, or acombination therapy sufficient to produce a desired therapeutic outcome(e.g., reducing the severity or duration of, stabilizing the severityof, or eliminating one or more symptoms of cancer). For therapeutic use,beneficial or desired results include, e.g., decreasing one or moresymptoms resulting from the disease (biochemical, histologic and/orbehavioral), including its complications and intermediate pathologicalphenotypes presenting during development of the disease, increasing thequality of life of those suffering from the disease, decreasing the doseof other medications required to treat the disease, enhancing effect ofanother medication, delaying the progression of the disease, and/orprolonging survival of patients.

A “prophylactically effective amount” refers to an amount of acomposition (e.g., nanoparticles that comprise rapamycin or a derivativethereof and a carrier protein), first therapy, second therapy, or acombination therapy sufficient to prevent or reduce the severity of oneor more future symptoms of cancer when administered to an individual whois susceptible and/or who may develop cancer. For prophylactic use,beneficial or desired results include, e.g., results such as eliminatingor reducing the risk, lessening the severity of future disease, ordelaying the onset of the disease (e.g., delaying biochemical,histologic and/or behavioral symptoms of the disease, its complications,and intermediate pathological phenotypes presenting during futuredevelopment of the disease).

The term “simultaneous administration,” as used herein, means that afirst therapy and second therapy in a combination therapy areadministered with a time separation of no more than about 15 minutes,such as no more than about any of 10, 5, or 1 minutes. When the firstand second therapies are administered simultaneously, the first andsecond therapies may be contained in the same composition (e.g., acomposition comprising both a first and second therapy) or in separatecompositions (e.g., a first therapy in one composition and a secondtherapy is contained in another composition).

As used herein, the term “sequential administration” means that thefirst therapy and second therapy in a combination therapy areadministered with a time separation of more than about 15 minutes, suchas more than about any of 20, 30, 40, 50, 60, or more minutes. Eitherthe first therapy or the second therapy may be administered first. Thefirst and second therapies are contained in separate compositions, whichmay be contained in the same or different packages or kits.

The term “proteins” refers to polypeptides or polymers of amino acids ofany length (including full length or fragments), which may be linear orbranched, comprise modified amino acids, and/or be interrupted bynon-amino acids. The term also encompasses an amino acid polymer thathas been modified naturally or by intervention, including, for example,disulfide bond formation, glycosylation, lipidation, acetylation,phosphorylation, or any other manipulation or modification. Alsoincluded within this term are, for example, polypeptides containing oneor more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Theproteins described herein may be naturally-occurring, i.e., obtained orderived from a natural source (e.g., blood) or synthesized (e.g.,chemically synthesized or by synthesized by recombinant DNA techniques).Exemplary carrier proteins are described herein.

The term “antimicrobial agent” used herein refers to an agent that iscapable of inhibiting (e.g., delaying, reducing, slowing, and/orpreventing) the growth of one or more microorganisms. Significantmicrobial growth can be measured or indicated by a number of ways knownin the art, such as one or more of the following: (i) microbial growthin a composition that is enough to cause one or more adverse effects toan individual when the composition is administered to the individual;(ii) more than about 10-fold increase in microbial growth over a certainperiod of time (for example over a 24 hour period) upon extrinsiccontamination (e.g., exposure to 10-103 colony forming units at atemperature in the range of 20 to 25° C.). Other indicia of significantmicrobial growth are described in U.S. Ser. No. 11/514,030, filed Aug.30, 2006, which is hereby incorporated by reference in its entirety.

“Sugar” as used herein includes, but is not limited to, monosaccharides,disaccharides, polysaccharides, and derivatives or modificationsthereof. Suitable sugars for compositions described herein include, forexample, mannitol, sucrose, fructose, lactose, maltose, and trehalose.

As used herein, by “pharmaceutically acceptable” or “pharmacologicallycompatible” is meant a material that is not biologically or otherwiseundesirable, e.g., the material may be incorporated into apharmaceutical composition administered to a patient without causing anysignificant undesirable biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained. Pharmaceutically acceptable carriers orexcipients have preferably met the required standards of toxicologicaland manufacturing testing and/or are included on the Inactive IngredientGuide prepared by the U.S. Food and Drug administration.

As used herein, reference to “not” a value or parameter generally meansand describes “other than” a value or parameter. For example, if ataxane is not administered, it means an agent other than a taxane isadministered.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise. It is understood that aspect and embodiments of the inventiondescribed herein include “consisting” and/or “consisting essentially of”aspects and embodiments.

Methods of Treating Cancer

The invention provides methods of treating cancer in an individual(e.g., human) comprising administering to the individual an effectiveamount of a composition comprising nanoparticles comprising rapamycin ora derivative thereof and a carrier protein (e.g., albumin). The presentinvention provides a method of treating cancer in an individual (e.g.,human) comprising administering to the individual an effective amount ofa composition comprising nanoparticles comprising rapamycin and analbumin. The therapy may be monotherapy or in a combination therapycontext. Additionally, the invention provides a method of treatingcancer in an individual by administering to the individual an effectiveamount of a combination of a) a first therapy that comprises acomposition comprising nanoparticles that comprise rapamycin or aderivative thereof and a carrier protein (e.g., albumin) and b) a secondtherapy useful for treating cancer. In some embodiments, the secondtherapy includes surgery, radiation, gene therapy, immunotherapy, bonemarrow transplantation, stem cell transplantation, hormone therapy,targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy,and/or chemotherapy (e.g., one or more compounds useful for treatingcancer). It is understood that reference to and description of methodsof treating cancer below is exemplary and that this description appliesequally to and includes methods of treating cancer using combinationtherapy.

Examples of cancers that may be treated by the methods of the inventioninclude, but are not limited to, adenocortical carcinoma, agnogenicmyeloid metaplasia, AIDS-related cancers (e.g., AIDS-related lymphoma),anal cancer, appendix cancer, astrocytoma (e.g., cerebellar andcerebral), basal cell carcinoma, bile duct cancer (e.g., extrahepatic),bladder cancer, bone cancer, (osteosarcoma and malignant fibroushistiocytoma), brain tumor (e.g., glioma, brain stem glioma, cerebellaror cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuseastrocytoma, anaplastic (malignant) astrocytoma), malignant glioma,ependymoma, oligodenglioma, meningioma, craniopharyngioma,haemangioblastomas, medulloblastoma, supratentorial primitiveneuroectodermal tumors, visual pathway and hypothalamic glioma, andglioblastoma), breast cancer, bronchial adenomas/carcinoids, carcinoidtumor (e.g., gastrointestinal carcinoid tumor), carcinoma of unknownprimary, central nervous system lymphoma, cervical cancer, colon cancer,colorectal cancer, chronic myeloproliferative disorders, endometrialcancer (e.g., uterine cancer), ependymoma, esophageal cancer, Ewing'sfamily of tumors, eye cancer (e.g., intraocular melanoma andretinoblastoma), gallbladder cancer, gastric (stomach) cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST),germ cell tumor, (e.g., extracranial, extragonadal, ovarian),gestational trophoblastic tumor, head and neck cancer, hepatocellular(liver) cancer (e.g., hepatic carcinoma and heptoma), hypopharyngealcancer, islet cell carcinoma (endocrine pancreas), laryngeal cancer,laryngeal cancer, leukemia, lip and oral cavity cancer, oral cancer,liver cancer, lung cancer (e.g., small cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung, and squamous carcinoma of thelung), lymphoid neoplasm (e.g., lymphoma), medulloblastoma, melanoma,mesothelioma, metastatic squamous neck cancer, mouth cancer, multipleendocrine neoplasia syndrome, myelodysplastic syndromes,myelodysplastic/myeloproliferative diseases, nasal cavity and paranasalsinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrinecancer, oropharyngeal cancer, ovarian cancer (e.g., ovarian epithelialcancer, ovarian germ cell tumor, ovarian low malignant potential tumor),pancreatic cancer, parathyroid cancer, penile cancer, cancer of theperitoneal, pharyngeal cancer, pheochromocytoma, pineoblastoma andsupratentorial primitive neuroectodermal tumors, pituitary tumor,pleuropulmonary blastoma, lymphoma, primary central nervous systemlymphoma (microglioma), pulmonary lymphangiomyomatosis, rectal cancer,renal cancer, renal pelvis and ureter cancer (transitional cell cancer),rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., non-melanoma(e.g., squamous cell carcinoma), melanoma, and Merkel cell carcinoma),small intestine cancer, squamous cell cancer, testicular cancer, throatcancer, thymoma and thymic carcinoma, thyroid cancer, tuberoussclerosis, urethral cancer, vaginal cancer, vulvar cancer, Wilms' tumor,and post-transplant lymphoproliferative disorder (PTLD), abnormalvascular proliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome.

In some embodiments, there are provided methods of treating cancer in anindividual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles comprisingrapamycin or a derivative thereof and a carrier protein (e.g., albumin),wherein the cancer is a lymphoid neoplasm (e.g., lymphoma). In someembodiments, there are provided methods of treating cancer in anindividual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles comprising arapamycin and an albumin, wherein the cancer is a lymphoid neoplasm(e.g., lymphoma).

In some embodiments the lymphoid neoplasm (e.g., lymphoma) is a B-cellneoplasm. Examples of B-cell neoplasms include, but are not limited to,precursor B-cell neoplasms (e.g., precursor B-lymphoblasticleukemia/lymphoma) and peripheral B-cell neoplasms (e.g., B-cell chroniclymphocytic leukemia/prolymphocytic leukemia/small lymphocytic lymphoma(small lymphocytic (SL) NHL), lymphoplasmacytoid lymphoma/immunocytoma,mantel cell lymphoma, follicle center lymphoma, follicular lymphoma(e.g., cytologic grades: I (small cell), II (mixed small and largecell), III (large cell) and/or subtype: diffuse and predominantly smallcell type), low grade/follicular non-Hodgkin's lymphoma (NHL),intermediate grade/follicular NHL, marginal zone B-cell lymphoma (e.g.,extranodal (e.g., MALT-type +/− monocytoid B cells) and/or Nodal (e.g.,+/− monocytoid B cells)), splenic marginal zone lymphoma (e.g., +/−villous lymphocytes), Hairy cell leukemia, plasmacytoma/plasma cellmyeloma (e.g., myeloma and multiple myeloma), diffuse large B-celllymphoma (e.g., primary mediastinal (thymic) B-cell lymphoma),intermediate grade diffuse NHL, Burkitt's lymphoma, High-grade B-celllymphoma, Burkitt-like, high grade immunoblastic NHL, high gradelymphoblastic NHL, high grade small non-cleaved cell NHL, bulky diseaseNHL, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia).

In some embodiments the lymphoid neoplasm (e.g., lymphoma) is a T-celland/or putative NK-cell neoplasm. Examples of T-cell and/or putativeNK-cell neoplasms include, but are not limited to, precursor T-cellneoplasm (precursor T-lymphoblastic lymphoma/leukemia) and peripheralT-cell and NK-cell neoplasms (e.g., T-cell chronic lymphocyticleukemia/prolymphocytic leukemia, and large granular lymphocyte leukemia(LGL) (e.g., T-cell type and/or NK-cell type), cutaneous T-cell lymphoma(e.g., mycosis fungoides/Sezary syndrome), primary T-cell lymphomasunspecified (e.g., cytological categories (e.g., medium-sized cell,mixed medium and large cell), large cell, lymphoepitheloid cell, subtypehepatosplenic γδ T-cell lymphoma, and subcutaneous panniculitic T-celllymphoma), angioimmunoblastic T-cell lymphoma (AILD), angiocentriclymphoma, intestinal T-cell lymphoma (e.g., +/− enteropathy associated),adult T-cell lymphoma/leukemia (ATL), anaplastic large cell lymphoma(ALCL) (e.g., CD30+, T- and null-cell types), anaplastic large-celllymphoma, and Hodgkin's like).

In some embodiments the lymphoid neoplasm (e.g., lymphoma) is Hodgkin'sdisease. For example, the Hodgkin's disease may be lymphocytepredominance, nodular sclerosis, mixed cellularity, lymphocytedepletion, and/or lymphocyte-rich.

In some embodiments, there are provided methods of treating cancer in anindividual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles comprisingrapamycin or a derivative thereof and a carrier protein (e.g., albumin),wherein the cancer is leukemia. In some embodiments, there are providedmethods of treating cancer in an individual by administering to theindividual (e.g., a human) an effective amount of a compositioncomprising nanoparticles comprising a rapamycin and an albumin, whereinthe cancer is leukemia. In some embodiments, the leukemia is chronicleukemia. Examples of chronic leukemia include, but are not limited to,chronic myelocytic I (granulocytic) leukemia, chronic myelogenous, andchronic lymphocytic leukemia (CLL). In some embodiments, the leukemia isacute leukemia. Examples of acute leukemia include, but are not limitedto, acute lymphoblastic leukemia (ALL), acute myeloid leukemia, acutelymphocytic leukemia, and acute myelocytic leukemia (e.g., myeloblastic,promyelocytic, myelomonocytic, monocytic, and erythroleukemia).

In some embodiments, there are provided methods of treating cancer withcompositions comprising nanoparticles comprising rapamycin or aderivative thereof and a carrier protein (e.g., albumin), wherein thecancer is a liquid tumor or plasmacytoma. In some embodiments, there areprovided methods of treating cancer in an individual by administering tothe individual (e.g., a human) an effective amount of a compositioncomprising nanoparticles comprising a rapamycin and an albumin, whereinthe cancer is a liquid tumor or plasmacytoma. Plasmacytoma includes, butis not limited to, myeloma. Myeloma includes, but is not limited to, anextramedullary plasmacytoma, a solitary myeloma, and multiple myeloma.In some embodiments, the plasmacytoma is multiple myeloma.

In some embodiments, there are provided methods of treating cancer in anindividual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles comprisingrapamycin or a derivative thereof and a carrier protein (e.g., albumin),wherein the cancer is multiple myeloma. In some embodiments, there areprovided methods of treating cancer in an individual by administering tothe individual (e.g., a human) an effective amount of a compositioncomprising nanoparticles comprising a rapamycin and an albumin, whereinthe cancer is multiple myeloma. Examples of multiple myeloma include,but are not limited to, IgG multiple myeloma, IgA multiple myeloma, IgDmultiple myeloma, IgE multiple myeloma, and nonsecretory multiplemyeloma. In some embodiments, the multiple myeloma is IgG multiplemyeloma. In some embodiments, the multiple myeloma is IgA multiplemyeloma. In some embodiments, the multiple myeloma is a smoldering orindolent multiple myeloma. In some embodiments, the multiple myeloma isprogressive multiple myeloma. In some embodiments, multiple myeloma maybe resistant to a drug, such as, but not limited to, bortezomib,dexamethasone (Dex-), doxorubicin (Dox-), and melphalan (LR).

In some embodiments, the individual may be a human who has a gene,genetic mutation, or polymorphism associated with multiple myeloma(e.g., ras, PTEN, Rb1, MTS1/p161NK4A/CDKN2, MTS2/p151NK4B, and/or p53)or has one or more extra copies of a gene associated with multiplemyeloma. In some embodiments, the individual has a ras or PTEN mutation.In some embodiments, the cancer cells are dependent on an mTOR pathwayto translate one or more mRNAs. In some embodiments, the cancer cellsare not capable of synthesizing mRNAs by an mTOR-independent pathway. Insome embodiments, the cancer cells have decreased or no PTEN activity orhave decreased or no expression of PTEN compared to non-cancerous cells.In some embodiments, the cancer cells have increased AKT activity and/orexpression compared to non-cancerous cells.

In some embodiments, there are provided methods of treating cancer in anindividual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles of rapamycinor a derivative thereof and a carrier protein (e.g., albumin), whereinthe cancer is a solid tumor. In some embodiments, there are providedmethods of treating cancer in an individual by administering to theindividual (e.g., a human) an effective amount of a compositioncomprising nanoparticles comprising a rapamycin and an albumin, whereinthe cancer is a solid tumor. In some embodiments, the solid tumorincludes, but is not limited to, sarcomas and carcinomas such asfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, Kaposi's sarcoma, soft tissue sarcoma,uterine sacronomasynovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Accordingly, in some embodiments, there are provided methods of treatingcancer in an individual by administering to the individual (e.g., ahuman) an effective amount of a composition comprising nanoparticlescomprising rapamycin or a derivative thereof and a carrier protein(e.g., albumin), wherein the cancer is breast cancer. In someembodiments, there are provided a method of treating breast cancer in anindividual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles that compriserapamycin or a derivative thereof and an albumin. In some embodiments,the breast cancer is early stage breast cancer, non-metastatic breastcancer, advanced breast cancer, stage 1V breast cancer, locally advancedbreast cancer, metastatic breast cancer, breast cancer in remission,breast cancer in an adjuvant setting, or breast cancer in a neoadjuvantsetting. In some specific embodiments, the breast cancer is in aneoadjuvant setting. In some embodiments, there are provided methods oftreating cancer at advanced stage(s). In some embodiments, there areprovided methods of treating breast cancer (which may be HER2 positiveor HER2 negative), including, for example, advanced breast cancer, stage1V breast cancer, locally advanced breast cancer, and metastatic breastcancer. In some embodiments, the individual may be a human who has agene, genetic mutation, or polymorphism associated with breast cancer(e.g., BRCA1, BRCA2, ATM, CHEK2, RAD51, AR, DIRAS3, ERBB2, TP53, AKT,PTEN, and/or PI3K) or has one or more extra copies of a gene (e.g., oneor more extra copies of the HER2 gene) associated with breast cancer. Insome embodiments, the method further comprises identifying a cancerpatient population (i.e. breast cancer population) based on a hormonereceptor status of patients having tumor tissue not expressing both ERand PgR and administering to the patient population an effective amountof a composition comprising nanoparticles comprising rapamycin or aderivative thereof and a carrier protein (e.g., albumin)

In some embodiments, there are provided methods of treating cancer in anindividual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles comprisingrapamycin or a derivative thereof and a carrier protein (e.g., albumin),wherein the cancer is a renal cell carcinoma (also called kidney cancer,renal adenocarcinoma, or hypernephroma). In some embodiments, there areprovided methods of treating cancer by administering to the individual(e.g., a human) an effective amount of a composition comprisingnanoparticles comprising an albumin, wherein the cancer is a renal cellcarcinoma. In some embodiments, the renal cell carcinoma is anadenocarcinoma. In some embodiments, the renal cell carcinoma is a clearcell renal cell carcinoma, papillary renal cell carcinoma (also calledchromophilic renal cell carcinoma), chromophobe renal cell carcinoma,collecting duct renal cell carcinoma, granular renal cell carcinoma,mixed granular renal cell carcinoma, renal angiomyolipomas, or spindlerenal cell carcinoma. In some embodiments, the individual may be a humanwho has a gene, genetic mutation, or polymorphism associated with renalcell carcinoma (e.g., VHL, TSC1, TSC2, CUL2, MSH2, MLH1, INK4a/ARF, MET,TGF-α, TGF-β1, IGF-1, IGF-1R, AKT, and/or PTEN) or has one or more extracopies of a gene associated with renal cell carcinoma. In someembodiments, the renal cell carcinoma is associated with (1) vonHippel-Lindau (VHL) syndrome, (2) hereditary papillary renal carcinoma(HPRC), (3) familial renal oncocytoma (FRO) associated withBirt-Hogg-Dube syndrome (BHDS), or (4) hereditary renal carcinoma (HRC).There are provided methods of treating renal cell carcinoma at any ofthe four stages, I, II, III, or IV, according to the American JointCommittee on Cancer (AJCC) staging groups. In some embodiments, therenal cell carcinoma is stage 1V renal cell carcinoma.

In some embodiments, there are provided methods of treating cancer in anindividual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles comprisingrapamycin or a derivative thereof and a carrier protein (e.g., albumin),wherein the cancer is prostate cancer. In some embodiments, there areprovided methods of treating cancer by administering to the individual(e.g., a human) an effective amount of a composition comprisingnanoparticles comprising a rapamycin and an albumin, wherein the canceris prostate cancer. In some embodiments, the prostate cancer is anadenocarcinoma. In some embodiments, the prostate cancer is a sarcoma,neuroendocrine tumor, small cell cancer, ductal cancer, or a lymphoma.There are provided methods of treating prostate cancer at any of thefour stages, A, B, C, or D, according to the Jewett staging system. Insome embodiments, the prostate cancer is stage A prostate cancer (Thecancer cannot be felt during a rectal exam.). In some embodiments; theprostate cancer is stage B prostate cancer (The tumor involves moretissue within the prostate, it can be felt during a rectal exam, or itis found with a biopsy that is done because of a high PSA level.). Insome embodiments, the prostate cancer is stage C prostate cancer (Thecancer has spread outside the prostate to nearby tissues.). In someembodiments, the prostate cancer is stage D prostate cancer. In someembodiments, the prostate cancer may be androgen independent prostatecancer (AIPC). In some embodiments, the prostate cancer may be androgendependent prostate cancer. In some embodiments, the prostate cancer maybe refractory to hormone therapy. In some embodiments, the prostatecancer may be substantially refractory to hormone therapy. In someembodiments, the individual may be a human who has a gene, geneticmutation, or polymorphism associated with prostate cancer (e.g.,RNASEL/HPC1, ELAC2/HPC2, SR-A1MSR1, CHEK2, BRCA2, PON1, OGG1, MIC-1,TLR4, and/or PTEN) or has one or more extra copies of a gene associatedwith prostate cancer.

In some embodiments, there are wprovided methods of treating cancer inan individual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles comprisingrapamycin or a derivative thereof and a carrier protein (e.g., albumin),wherein the cancer is lung cancer. In some embodiments, there areprovided methods of treating cancer by administering to the individual(e.g., a human) an effective amount of a composition comprisingnanoparticles comprising a rapamycin and an albumin, wherein the canceris lung cancer. In some embodiments, the cancer is lung cancer is anon-small cell lung cancer (NSCLC). Examples of NCSLC include, but arenot limited to, large-cell carcinoma (e.g., large-cell neuroendocrinecarcinoma, combined large-cell neuroendocrine carcinoma, basaloidcarcinoma, lymphoepithelioma-like carcinoma, clear cell carcinoma, andlarge-cell carcinoma with rhabdoid phenotype), adenocarcinoma (e.g.,acinar, papillary (e.g., bronchioloalveolar carcinoma, nonmucinous,mucinous, mixed mucinous and nonmucinous and indeterminate cell type),solid adenocarcinoma with mucin, adenocarcinoma with mixed subtypes,well-differentiated fetal adenocarcinoma, mucinous (colloid)adenocarcinoma, mucinous cystadenocarcinoma, signet ring adenocarcinoma,and clear cell adenocarcinoma), neuroendocrine lung tumors, and squamouscell carcinoma (e.g., papillary, clear cell, small cell, and basaloid).In some embodiments, the NSCLC may be, according to TNM classifications,a stage T tumor (primary tumor), a stage N tumor (regional lymph nodes),or a stage M tumor (distant metastasis). In some embodiments, the lungcancer is a carcinoid (typical or atypical), adenosquamous carcinoma,cylindroma, or carcinoma of the salivary gland (e.g., adenoid cysticcarcinoma or mucoepidermoid carcinoma). In some embodiments, the lungcancer is a carcinoma with pleomorphic, sarcomatoid, or sarcomatouselements (e.g., carcinomas with spindle and/or giant cells, spindle cellcarcinoma, giant cell carcinoma, carcinosarcoma, or pulmonary blastoma).In some embodiments, the cancer is small cell lung cancer (SCLC; alsocalled oat cell carcinoma). The small cell lung cancer may belimited-stage, extensive stage or reoccurent small cell lung cancer. Insome embodiments, the individual may be a human who has a gene, geneticmutation, or polymorphism suspected or shown to be associated with lungcancer (e.g., SASH1, LATS1, IGF2R, PARK2, KRAS, PTEN, Kras2, Krag, Pas1,ERCC1, XPD, IL8RA, EGFR, α₁-AD, EPHX, MMP1, MMP2, MMP3, MMP12, IL1β,RAS, and/or AKT) or has one or more extra copies of a gene associatedwith lung cancer.

In some embodiments, there are provided methods of treating cancer in anindividual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles comprisingrapamycin or a derivative thereof and a carrier protein (e.g., albumin),wherein the cancer is brain cancer. In some embodiments, there areprovided methods of treating cancer by administering to the individual(e.g., a human) an effective amount of a composition comprisingnanoparticles comprising a rapamycin and an albumin, wherein the canceris brain cancer. In some embodiments, the brain cancer is glioma, brainstem glioma, cerebellar or cerebral astrocytoma (e.g., pilocyticastrocytoma, diffuse astrocytoma, or anaplastic (malignant)astrocytoma), malignant glioma, ependymoma, oligodenglioma, meningioma,craniopharyngioma, haemangioblastomas, medulloblastoma, supratentorialprimitive neuroectodermal tumors, visual pathway and hypothalamicglioma, or glioblastoma. In some embodiments, the brain cancer isglioblastoma (also called glioblastoma multiforme or grade 4astrocytoma). In some embodiments, the glioblastoma isradiation-resistant. In some embodiments, the glioblastoma isradiation-sensitive. In some embodiments, the glioblastoma may beinfratentorial. In some embodiments, the glioblastoma is supratentorial.In some embodiments, the individual may be a human who has a gene,genetic mutation, or polymorphism associated with brain cancer (e.g.,glioblastoma) (e.g., NRPB, MAGE-E1, MMACI-E1, PTEN, LOH, p53, MDM2, DCC,TP-73, Rb1, EGFR, PDGFR-α, PMS2, MLH1, and/or DMBT1) or has one or moreextra copies of a gene associated with brain cancer (e.g., glioblastoma)(e.g., MDM2, EGFR, and PDGR-α).

In some embodiments, there are provided methods of treating cancer in anindividual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles comprisingrapamycin or a derivative thereof and a carrier protein (e.g., albumin),wherein the cancer is melanoma. In some embodiments, there are providedmethods of treating cancer by administering to the individual (e.g., ahuman) an effective amount of a composition comprising nanoparticlescomprising a rapamycin and an albumin, wherein the cancer is melanoma.

In some embodiments, there are provided methods of treating cancer in anindividual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles comprisingrapamycin or a derivative thereof and a carrier protein (e.g., albumin),wherein the cancer is ovarian cancer. In some embodiments, there areprovided methods of treating cancer by administering to the individual(e.g., a human) an effective amount of a composition comprisingnanoparticles comprising a rapamycin and an albumin, wherein the canceris ovarian cancer. In some embodiments, the cancer is ovarian epithelialcancer. Exemplary ovarian epithelial cancer histological classificationsinclude: serous cystomas (e.g., serous benign cystadenomas, serouscystadenomas with proliferating activity of the epithelial cells andnuclear abnormalities but with no infiltrative destructive growth, orserous cystadenocarcinomas), mucinous cystomas (e.g., mucinous benigncystadenomas, mucinous cystadenomas with proliferating activity of theepithelial cells and nuclear abnormalities but with no infiltrativedestructive growth, or mucinous cystadenocarcinomas), endometrioidtumors (e.g., endometrioid benign cysts, endometrioid tumors withproliferating activity of the epithelial cells and nuclear abnormalitiesbut with no infiltrative destructive growth, or endometrioidadenocarcinomas), clear cell (mesonephroid) tumors (e.g., begin clearcell tumors, clear cell tumors with proliferating activity of theepithelial cells and nuclear abnormalities but with no infiltrativedestructive growth, or clear cell cystadenocarcinomas), unclassifiedtumors that cannot be allotted to one of the above groups, or othermalignant tumors. In various embodiments, the ovarian epithelial canceris stage I (e.g., stage IA, IB, or IC), stage II (e.g., stage IIA, IIB,or IIC), stage III (e.g., stage IIIA, IIIB, or IIIC), or stage IV. Insome embodiments, the individual may be a human who has a gene, geneticmutation, or polymorphism associated with ovarian cancer (e.g., BRCA1 orBRCA2) or has one or more extra copies of a gene associated with ovariancancer (e.g., one or more extra copies of the HER2 gene).

In some embodiments, the cancer is an ovarian germ cell tumor. Exemplaryhistologic subtypes include dysgerminomas or other germ cell tumors(e.g., endodermal sinus tumors such as hepatoid or intestinal tumors,embryonal carcinomas, olyembryomas, choriocarcinomas, teratomas, ormixed form tumors). Exemplary teratomas are immature teratomas, matureteratomas, solid teratomas, and cystic teratomas (e.g., dermoid cystssuch as mature cystic teratomas, and dermoid cysts with malignanttransformation). Some teratomas are monodermal and highly specialized,such as struma ovarii, carcinoid, struma ovarii and carcinoid, or others(e.g., malignant neuroectodermal and ependymomas). In some embodiments,the ovarian germ cell tumor is stage I (e.g., stage IA, IB, or IC),stage II (e.g., stage IIA, IIB, or IIC), stage III (e.g., stage IIIA,IIIB, or IIIC), or stage IV.

In some embodiments, there are provided methods of treating cancer in anindividual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles comprisingrapamycin or a derivative thereof and a carrier protein (e.g., albumin),wherein the cancer is a neuroendocrine cancer. In some embodiments, theindividual may be a human who has a gene, genetic mutation, orpolymorphism associated with neuroendocrine cancer (e.g., TSC1, TSC2,IGF-1, IGF-1R, and/or VHL) or has one or more extra copies of a geneassociated with neurondocrine cancer.

In some embodiments, there are provided methods of treating cancer in anindividual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles comprisingrapamycin or a derivative thereof and a carrier protein (e.g., albumin),wherein the cancer is colon cancer. In some embodiments, the individualmay be a human who has a gene, genetic mutation, or polymorphismassociated with colon cancer (e.g., RAS, AKT, PTEN, PI3K, and/or EGFR)or has one or more extra copies of a gene associated with colon cancer.

In some embodiments, there are provided methods of treating cancer in anindividual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles comprisingrapamycin or a derivative thereof and a carrier protein (e.g., albumin),wherein the cancer is characterized by PI3K and/or AKT activation. Insome embodiments, the cancer characterized by PI3K and/or AKT activationis HER2⁺ breast cancer, chronic myelogenous leukemia, ovarian cancer,endometrial cancer, sarcoma, squamous cell carcinoma of the head andneck, or thyroid cancer. In some variations, the cancer is furthercharacterized by AKT gene amplification.

In some embodiments, there are provided methods of treating cancer in anindividual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles comprisingrapamycin or a derivative thereof and a carrier protein (e.g., albumin),wherein the cancer is characterized by cyclin D1 overexpression. In someembodiments, the cancer characterized by cyclin D overexpression ismantle cell lymphoma or breast cancer.

In some embodiments, there are provided methods of treating cancer in anindividual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles comprisingrapamycin or a derivative thereof and a carrier protein (e.g., albumin),wherein the cancer is characterized by cMYC overexpression. In someembodiments, the cancer characterized by cMYC overexpression is Burkittlymphoma.

In some embodiments, there are provided methods of treating cancer in anindividual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles comprisingrapamycin or a derivative thereof and a carrier protein (e.g., albumin),wherein the cancer is characterized by HIF overexpression. In someembodiments, the cancer characterized by HIF overexpression is renalcell carcinoma or Von Hippel-Lindau. In some embodiments, the cancerfurther comprises a VHL mutation.

In some embodiments, there are provided methods of treating cancer in anindividual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles comprisingrapamycin or a derivative thereof and a carrier protein (e.g., albumin),wherein the cancer is characterized by TSC1 and/or TSC2 loss. In someembodiments, the cancer characterized by TSC 1 and/or TSC2 is tuberoussclerosis or pulmonary lymphangiomyomatosis.

In some embodiments, there are provided methods of treating cancer in anindividual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles comprisingrapamycin or a derivative thereof and a carrier protein (e.g., albumin),wherein the cancer is characterized by a TSC2 mutation. In someembodiments, the cancer characterized by TSC2 mutation is renalangiomyolipomas.

In some embodiments, there are provided methods of treating cancer in anindividual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles comprisingrapamycin or a derivative thereof and a carrier protein (e.g., albumin),wherein the cancer is characterized by a PTEN mutation. In someembodiments, the PTEN mutation is a loss of PTEN function. In someembodiments, the cancer characterized by a PTEN mutation isglioblastoma, endometrial cancer, prostate cancer, sarcoma, or breastcancer.

In some embodiments, the methods of treatment provided herein may alsobe used to treat a cancer which is not a solid tumor (i.e., other than asolid tumor). In some embodiments, the methods of treatment providedherein may also be used to treat a cancer which is not a carcinoma. Insome embodiments, the methods of treatment provided herein may also beused to treat a cancer which is not a sarcoma. In some embodiments, themethods of treatment provided herein may also be used to treat a cancerwhich is not a lymphoma. In some embodiments, the methods of treatmentprovided herein may also be used to treat a cancer which is not coloncancer (i.e., other than colon cancer). In some embodiments, the methodsof treatment provided herein may also be used to treat a cancer which isnot breast cancer (i.e., other than breast cancer). In some embodiments,the methods of treatment provided herein may also be used to treat acancer which is not an ovarian cancer, brain cancer, and/or prostatecancer (i.e., other than ovarian cancer, brain cancer, and/or prostatecancer).

Any of the methods of treatment provided herein may be used to treat aprimary tumor. Any of the methods of treatment provided herein may alsobe used to treat a metastatic cancer (that is, cancer that hasmetastasized from the primary tumor). Any of the methods of treatmentprovided herein may be used to treat cancer at an advanced stage. Any ofthe methods of treatment provided herein may be used to treat cancer atlocally advanced stage. Any of the methods of treatment provided hereinmay be used to treat early stage cancer. Any of the methods of treatmentprovided herein may be used to treat cancer in remission. In some of theembodiments of any of the methods of treatment provided herein, thecancer has reoccurred after remission. In some embodiments of any of themethods of treatment provided herein, the cancer is progressive cancer.Any of the methods of treatment provided herein may be used to treatcancer substantially refractory to hormone therapy. Any of the methodsof treatment provided herein may be used to treat HER-2 positive cancer.Any of the methods of treatment provided herein may be used to treatHER-2 negative cancer.

Any of the methods of treatment provided herein may be used to treat andindividual (e.g., human) who has been diagnosed with or is suspected ofhaving cancer. In some embodiments, the individual may be a human whoexhibits one or more symptoms associated with cancer. In someembodiments, the individual may have advanced disease or a lesser extentof disease, such as low tumor burden. In some embodiments, theindividual is at an early stage of a cancer. In some embodiments, theindividual is at an advanced stage of cancer. In some of the embodimentsof any of the methods of treatment provided herein, the individual maybe a human who is genetically or otherwise predisposed (e.g., riskfactor) to developing cancer who has or has not been diagnosed withcancer. In some embodiments, these risk factors include, but are notlimited to, age, sex, race, diet, history of previous disease, presenceof precursor disease, genetic (e.g., hereditary) considerations, andenvironmental exposure. In some embodiments, the individuals at risk forcancer include, e.g., those having relatives who have experienced thisdisease, and those whose risk is determined by analysis of genetic orbiochemical markers.

Any of the methods of treatment provided herein may be practiced in anadjuvant setting. Any of the methods of treatment provided herein may bepracticed in a neoadjuvant setting, i.e., the method may be carried outbefore the primary/definitive therapy. In some embodiments, any of themethods of treatment provided herein may be used to treat an individualwho has previously been treated. Any of the methods of treatmentprovided herein may be used to treat an individual who has notpreviously been treated. Any of the methods of treatment provided hereinmay be used to treat an individual at risk for developing cancer, buthas not been diagnosed with cancer. Any of the methods of treatmentprovided herein may be used as a first line therapy. Any of the methodsof treatment provided herein may be used as a second line therapy.

In some embodiments of any of the methods of treatment provided herein,a taxane is not administered to the individual. In some embodiments, thetaxane administered is not a nanoparticle composition. In someembodiments, the nanoparticle composition comprising rapamycin or aderivative thereof is not administered in conjunction with a taxane. Insome embodiments, a taxane is not administered to the individual duringthe time period in which the individual is receiving one or more dosesof a nanoparticle composition comprising rapamycin or a derivativethereof. In some embodiments, the individual was treated with a taxanebefore treatment begins with a nanoparticle composition comprisingrapamycin or a derivative thereof. For example, the individual may havereceived a taxane one or more days, weeks, months, or years beforetreatment begins with a nanoparticle composition comprising rapamycin ora derivative thereof. In other embodiments, the individual neverreceives a taxane before treatment begins with a nanoparticlecomposition comprising rapamycin or a derivative thereof. In someembodiments, the individual is treated with a taxane after treatmentwith a nanoparticle composition comprising rapamycin or derivativethereof terminates. In other embodiments, the individual is nevertreated with a taxane after treatment with a nanoparticle compositioncomprising rapamycin or derivative thereof terminates. In someembodiments, the composition, first therapy, and/or second therapy donot contain a taxane. In other embodiments, the composition, firsttherapy, and/or second therapy comprise a taxane. In some embodiments,the first and/or second therapies do not comprise a SPARC polypeptide oranti-SPARC antibody (i.e., other than SPARC polypeptide or anti-SPARCantibody).

Any of the methods of treatment provided herein may be used to treat,stabilize, prevent, and/or delay any type or stage of cancer. In someembodiments, the individual is at least about any of 40, 45, 50, 55, 60,65, 70, 75, 80, or 85 years old. In some embodiments, one or moresymptoms of the cancer are ameliorated or eliminated. In someembodiments, the size of a tumor, the number of cancer cells, or thegrowth rate of a tumor decreases by at least about any of 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%. In some embodiments, thecancer is delayed or prevented.

Combination Therapy

The present invention also features methods for the treatment of cancerusing combination therapies. Accordingly, in some embodiments, a secondtherapy useful for treating cancer is also administered to theindividual. In some embodiments, the second therapy includes surgery,radiation, gene therapy, immunotherapy, bone marrow transplantation,stem cell transplantation, hormone therapy, targeted therapy,cryotherapy, ultrasound therapy, photodynamic therapy, and/orchemotherapy (e.g., one or more compounds or pharmaceutically acceptablesalts thereof useful for treating cancer). It is understood thatreference to and description of methods of treating cancer above isexemplary and that the description applies equally to and includesmethods of treating cancer using combination therapy.

In one such aspect, the invention provides a method of treating cancerin an individual by administering to the individual an effective amountof a combination of a) a first therapy that includes a compositioncomprising nanoparticles that include rapamycin or a derivative thereofand a carrier protein (e.g., albumin) and b) a second therapy useful fortreating cancer. In some embodiments, the second therapy includessurgery, radiation, gene therapy, immunotherapy, bone marrowtransplantation, stem cell transplantation, hormone therapy, targetedtherapy, cryotherapy, ultrasound therapy, photodynamic therapy, and/orchemotherapy (e.g., one or more compounds useful for treating cancer).In some embodiments, the first and/or second therapies do not include ataxane. In other embodiments, the first and/or second therapies doinclude a taxane. In some embodiments, the first and/or second therapiesdo not comprise a SPARC polypeptide or anti-SPARC antibody.

In some embodiments, the invention provides methods of treating cancerin an individual, comprising administering to the individual a) aneffective amount of a composition comprising nanoparticles comprising arapamycin or a derivative thereof and a carrier protein (e.g., albumin);and b) an effective amount of at least one other chemotherapeutic agent.In some embodiments, the nanoparticles comprise rapamycin and analbumin. In some embodiments, the chemotherapeutic agent is any of (andin some embodiments selected from the group consisting of)antimetabolite agents (including nucleoside analogs), platinum-basedagents, alkylating agents, tyrosine kinase inhibitors, anthracyclineantibiotics, vinca alkloids, proteasome inhibitors, taxanes, modulatorsof HER2/neu (such as inhibitors of HER2/neu for example Herceptin®),modulators of EGFR (such as inhibitors of EGFR for example Erbitux®),modulators of VEGFR, farnosyltransferase inhibitors, and topoisomeraseinhibitors. In some embodiments, the chemotherapeutic is not a taxane(i.e., the compound is a chemotherapeutic agent other than a taxane).Preferred drug combinations for sequential or co-administration orsimultaneous administration with nanoparticles comprising a rapamycin ora derivative thereof and a carrier protein (e.g., albumin) are thosewhich show enhanced anticancer activity when compared with the singlecomponents alone, especially combinations that lead to regression ofcancer and/or cure from cancer.

The chemotherapeutic agents described herein can be the agentsthemselves, pharmaceutically acceptable salts thereof, andpharmaceutically acceptable esters thereof, as well as stereoisomers,enantiomers, racemic mixtures, and the like. The chemotherapeutic agentor agents as described can be administered as well as a pharmaceuticalcomposition containing the agent(s), wherein the pharmaceuticalcomposition comprises a pharmaceutically acceptable carrier vehicle, orthe like.

The chemotherapeutic agent may be present in a nanoparticle composition.For example, in some embodiments, there is provided a method of treatingcancer in an individual, comprising administering to the individual a)an effective amount of a composition comprising nanoparticles comprisinga rapamycin or a derivative thereof and a carrier protein (e.g.,albumin); and b) an effective amount of a composition comprisingnanoparticles comprising at least one other chemotherapeutic agent and acarrier protein (such as albumin). In some embodiments, the methodcomprises administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising a rapamycin and analbumin; and b) an effective amount of a composition comprisingnanoparticles comprising at least one other chemotherapeutic agent and acarrier protein (such as albumin). In some embodiments, thechemotherapeutic agent is any of (and in some embodiments selected fromthe group consisting of) thiocolchicine or its derivatives (such asdimeric thiocolchicine, including for example nab-5404, nab-5800, andnab-5801), and geldanamycin or its derivatives (such as 17-allyl aminogeldanamycin (17-AAG)). In some embodiments, the chemotherapeutic agentis a taxane or a derivative thereof (e.g., paclitaxel, docetaxel, andortataxel). In some embodiments, the chemotherapeutic agent is not ataxane. In other embodiments, the chemotherapeutic is not a taxane. Insome embodiments, the chemotherapeutic agent is 17-AAG. In someembodiments, the chemotherapeutic agent is dimeric thiocolchicine.

An exemplary and non-limiting list of chemotherapeutic agentscontemplated is provided herein. Suitable chemotherapeutic agentsinclude, for example, vinca alkaloids, agents that disrupt microtubuleformation (such as colchicines and its derivatives), anti-angiogenicagents, therapeutic antibodies, EGFR targeting agents, tyrosine kinasetargeting agent (such as tyrosine kinase inhibitors), transitional metalcomplexes, proteasome inhibitors, antimetabolites (such as nucleosideanalogs), alkylating agents, platinum-based agents, anthracyclineantibiotics, topoisomerase inhibitors, therapeutic antibodies, retinoids(such as all-trans retinoic acids or a derivatives thereof);geldanamycin or a derivative thereof (such as 17-AAG), and otherstandard chemotherapeutic agents well recognized in the art.

In some embodiments, the chemotherapeutic agent is any of (and in someembodiments selected from the group consisting of) adriamycin,colchicine, cyclophosphamide, actinomycin, bleomycin, duanorubicin,doxorubicin, epirubicin, mitomycin, methotrexate, mitoxantrone,fluorouracil, carboplatin, carmustine (BCNU), methyl CCNU, cisplatin,etoposide, epotetin alfa, interferons (e.g., IFN-α), camptothecin andderivatives thereof, letrozole, panitumumab (Vectibix®), phenesterine,topetecan, vinblastine, vincristine, tamoxifen, thalidomide, tipifarnib(Zarnestra®), piposulfan, nab-5404, nab-5800, nab-5801, Irinotecan, HKP,Ortataxel, gemcitabine, Herceptin®, vinorelbine, Doxil®, capecitabine,Alimta®, Avastin®, Velcade®, Tarceva®, Neulasta®, Lapatinib, Sorafenib,derivatives thereof, chemotherapeutic agents known in the art, and thelike. In some embodiments, the chemotherapeutic agent is a compositioncomprising nanoparticles comprising a thiocolchicine derivative and acarrier protein (such as albumin). In some embodiments, thechemotherapeutic agent is a taxane or a derivative thereof (e.g.,paclitaxel, docetaxel, and ortataxel). In some embodiments, thechemotherapeutic is not a taxane.

In some embodiments, the chemotherapeutic agent is a antineoplasticagent including, but is not limited to, carboplatin, Navelbine®(vinorelbine), anthracycline (Doxil®), lapatinib (GW57016), Herceptin®,gemcitabine (Gemzar®), capecitabine (Xeloda®), Alimta®, cisplatin,5-fluorouracil, epirubicin, cyclophosphamide, Avastin®, Velcade®, etc.

In some embodiments, the chemotherapeutic agent is an antagonist ofother factors that are involved in tumor growth, such as EGFR, ErbB2(also known as Herb), ErbB3, ErbB4, or TNF. Sometimes, it may bebeneficial to also administer one or more cytokines to the individual.In some embodiments, the therapeutic agent is a growth inhibitory agent.Suitable dosages for the growth inhibitory agent are those presentlyused and may be lowered due to the combined action (synergy) of thegrowth inhibitory agent and the rapamycin or derivative thereof. In someembodiments, the chemotherapeutic agent is a chemotherapeutic agentother than an anti-VEGF antibody, a HER2 antibody, interferon, and anHGFβ antagonist.

Reference to a chemotherapeutic agent herein applies to thechemotherapeutic agent or its derivatives and accordingly the inventioncontemplates and includes either of these embodiments (agent; agent orderivative(s)). “Derivatives” of a chemotherapeutic agent or otherchemical moiety include, but are not limited to, compounds that arestructurally similar to the chemotherapeutic agent or moiety, compoundsthat are in the same general chemical class as the chemotherapeuticagent or moiety, analogs of chemotherapeutic agents, or pharmaceuticallyacceptable salts of chemotherapeutic agents or their derivatives. Insome embodiments, the derivative of the chemotherapeutic agent or moietyretains similar chemical and/or physical property (including, forexample, functionality) of the chemotherapeutic agent or moiety.

In some embodiments, the invention provides a method of treating cancerin an individual, comprising administering to the individual a) aneffective amount of a composition comprising nanoparticles comprising arapamycin or a derivative thereof and a carrier protein (e.g., albumin),and b) an effective amount of a tyrosine kinase inhibitor. In someembodiments, the invention provides a method of treating cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising rapamycinand an albumin, and b) an effective amount of a tyrosine kinaseinhibitor. Suitable tyrosine kinase inhibitors include, for example,imatinib (Gleevec®), nilotinim, gefitinib (Iressa®; ZD-1839), erlotinib(Tarceva®; OSI-774), sunitinib malate (Sutent®), sorafenib (Nexavar®),and Lapatinib (GW562016; Tykerb). In some embodiments, the tyrosinekinase inhibitor is a multiple reversible ErbB1 family tyrosine kinaseinhibitor (e.g., laptinib). In some embodiments, the tyrosine kinaseinhibitor is a single reversible EGFR tyrosine kinase inhibitor (e.g.,gefitinib or erlotinib). In some embodiments, the tyrosine kinaseinhibitor is erlotinib. In some embodiments, the tyrosine kinaseinhibitor is gefitinib. In some embodiments, the tyrosine kinaseinhibitor is a single irreversible EGFR tyrosine kinase inhibitor (e.g.,EKB-569 or CL-387,785). In some embodiments, the tyrosine kinaseinhibitor is a multiple irreversible ErbB family tyrosine kinaseinhibitor (e.g. canertinib (CL-1033; PD183805), HKI-272, BIBW 2992, orHKI-357). In some embodiments, the tyrosine kinase inhibitor is amultiple reversible tyrosine kinase inhibitor (e.g., ZD-6474, ZD-6464,AEE 788, or XL647). In some embodiments, the tyrosine kinase inhibitorinhibits ErbB family heterodimerization (e.g., BMS-599626). In someembodiments, the tyrosine kinase inhibitor inhibits protein folding byaffecting HSP90 (e.g., benzoquinone ansamycin, IPI-504, or 17-AAG). Insome embodiments, there is provided a method to inhibit theproliferation of EGFR expressing tumors in a mammal comprisingadministering to a mammal infected with such tumors an effective amountof a composition comprising nanoparticles comprising a rapamycin or aderivative thereof and a carrier protein (e.g., albumin) and gefitinib,wherein the gefitinib is administered by pulse-dosing. In someembodiments, the tyrosine kinase inhibitor is an inhibitor of BCR-Abl.In some embodiments, the tyrosine kinase inhibitor is an inhibitor ofIGF-1R.

In some embodiments, the method is for treating non-small cell lungcarcinoma. In some embodiments, the method is for treating brain cancer(e.g., glioblastoma). In some embodiments, the method is for treatingcolorectal cancer, gastrointestinal stromal tumor, prostate cancer,ovarian cancer, or thyroid cancer. In some embodiments, the method isfor treatment of prostate cancer (e.g., advanced prostate cancer). Insome embodiments, the method is for treatment of breast cancer,including treatment of metastatic breast cancer and treatment of breastcancer in a neoadjuvant setting. In some embodiments, the method is fortreatment of advanced solid tumor. In some embodiments, the method isfor treatment of multiple myeloma. In some embodiments, the methodcomprises simultaneous and/or sequential administration of at least oneEGFR blocker, inhibitor, or antagonist. In some embodiments, theindividual has activating mutation(s) in the kinase domain of EGFR. Insome embodiments, the individual is of Asian or East Asian ancestry. Insome embodiments, the individual is female.

In some embodiments, the invention provides a method of treating cancerin an individual, comprising administering to the individual a) aneffective amount of a composition comprising nanoparticles comprising arapamycin or a derivative thereof and a carrier protein (e.g., albumin),and b) an effective amount of an antimetabolite agent (such as anucleoside analog, including for example purine analogs and pyrimidineanalogs). In some embodiments, the invention provides a method oftreating cancer in an individual, comprising administering to theindividual a) an effective amount of a composition comprisingnanoparticles comprising rapamycin and an albumin, and b) an effectiveamount of an antimetabolite agent. An “antimetabolic agent” is an agentwhich is structurally similar to a metabolite, but cannot be used by thebody in a productive manner. Many antimetabolite agents interfere withproduction of nucleic acids, RNA and DNA. For example, theantimetabolite can be a nucleoside analog, which includes, but is notlimited to, azacitidine, azathioprine, capecitabine (Xeloda®),cytarabine, cladribine, cytosine arabinoside (ara-C, cytosar),doxifluridine, fluorouracil (such as 5-fluorouracil),9-(2-phosphonylmethoxyethyl)adenine, UFT, hydroxyurea, gemcitabine,mercaptopurine, methotrexate, thioguanine (such as 6-thioguanine). Otheranti-metabolites include, for example, L-asparaginase (Elspa),decarbazine (DTIC), 2-deoxy-D-glucose, and procarbazine (matulane). Insome embodiments, the nucleoside analog is any of (and in someembodiments selected from the group consisting of) gemcitabine,fluorouracil, and capecitabine. In some embodiments, the method is fortreatment of metastatic breast cancer or locally advanced breast cancer.In some embodiments, the method is for first line treatment ofmetastatic breast cancer. In some embodiments, the method is fortreatment of breast cancer in a neoadjuvant setting. In someembodiments, the method is for treatment of any of NSCLC, metastaticcolorectal cancer, pancreatic cancer, or advanced solid tumor.

In some embodiments, the invention provides a method of treating cancerin an individual, comprising administering to the individual a) aneffective amount of a composition comprising nanoparticles comprising arapamycin or a derivative thereof and a carrier protein (e.g., albumin),and b) an effective amount of an alkylating agent. In some embodiments,the invention provides a method of treating cancer in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising rapamycin and analbumin, and b) an effective amount of an alkylating agent. Suitablealkylating agents include, but are not limited to, cyclophosphamide(Cytoxan), mechlorethamine, chlorambucil, melphalan, carmustine (BCNU),thiotepa, busulfan, alkyl sulphonates, ethylene imines, nitrogen mustardanalogs, estramustine sodium phosphate, ifosfamide, nitrosoureas,lomustine, and streptozocin. In some embodiments, the alkylating agentis cyclophosphamide. In some embodiments, the cyclophosphamide isadministered prior to the administration of the nanoparticlecomposition. In some embodiments, the method is for treatment of anearly stage breast cancer. In some embodiments, the method is fortreatment of a breast cancer in an adjuvant or a neoadjuvant setting.

In some embodiments, the invention provides a method of treating cancerin an individual, comprising administering to the individual a) aneffective amount of a composition comprising nanoparticles comprising arapamycin or a derivative thereof and a carrier protein (e.g., albumin),and b) an effective amount of a platinum-based agent. In someembodiments, the invention provides a method of treating cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising rapamycinand an albumin, and b) an effective amount of a platinum-based agent.Suitable platinum-based agents include, but are not limited to,carboplatin, cisplatin, and oxaliplatin. In some embodiments, theplatinum-based agent is carboplatin. In some embodiments, theplatinum-based agent is oxaliplatin. We have observed that rapamycininhibited oxaliplatin induced apoptosis in a dose dependent manner. Thisinhibition was not overwhelmed by increasing amount of oxaliplatin up to1:1 (w/w) ratio of the two drugs. The same was observed for Eloxatin®(oxaliplatin injection).

In some embodiments, the method is for treatment of breast cancer (HER2positive or HER2 negative, including metastatic breast cancer andadvanced breast cancer); lung cancer (including advanced NSCLC, firstline NSCLC, SCLC, and advanced solid tumor malignancies in the lung);ovarian cancer; head and neck cancer; and melanoma (including metastaticmelanoma).

In some embodiments, the invention provides a method of treating cancerin an individual, comprising administering to the individual a) aneffective amount of a composition comprising nanoparticles comprising arapamycin or a derivative thereof and a carrier protein (e.g., albumin),and b) an effective amount of an anthracycline antibiotic. In someembodiments, the invention provides a method of treating cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising rapamycinand an albumin, and b) an effective amount of an anthracyclineantibiotic. Suitable anthracycline antibiotic include, but are notlimited to, Doxil®, actinomycin, dactinomycin, daunorubicin(daunomycin), doxorubicin (adriamycin), epirubicin, idarubicin,mitoxantrone, and valrubicin. In some embodiments, the anthracycline isany of (and in some embodiments selected from the group consisting of)Doxil®, epirubicin, and doxorubicin. In some embodiments, the method isfor treatment of an early stage breast cancer. In some embodiments, themethod is for treatment of a breast cancer in an adjuvant or aneoadjuvant setting.

In some embodiments, the invention provides a method of treating cancerin an individual, comprising administering to the individual a) aneffective amount of a composition comprising nanoparticles comprising arapamycin or a derivative thereof and a carrier protein (e.g., albumin),and b) an effective amount of a vinca alkloid. In some embodiments, theinvention provides a method of treating cancer in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising rapamycin and analbumin, and b) an effective amount of a vinca alkloid. Suitable vincaalkaloids include, for example, vinblastine, vincristine, vindesine,vinorelbine (Navelbine®), and VP-16. In some embodiments, the vincaalkaloid is vinorelbine (Navelbine®). In some embodiments, the method isfor treatment of stage IV breast cancer and lung cancer.

In some embodiments, the invention provides a method of treating cancerin an individual, comprising administering to the individual a) aneffective amount of a composition comprising nanoparticles comprising arapamycin or a derivative thereof and a carrier protein (e.g., albumin),and b) an effective amount of a topoisomerase inhibitor. In someembodiments, the invention provides a method of treating cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising rapamycinand an albumin, and b) an effective amount of a topoisomerase inhibitor.In some embodiments, the chemotherapeutic agent is a topoisomeraseinhibitor, including, for example, inhibitor of topoisomerase I andtopoisomerase II. Exemplary inhibitors of topoisomerase I include, butare not limited to, camptothecin, such as irinotecan and topotecan.Exemplary inhibitors of topoisomerase II include, but are not limitedto, amsacrine, etoposide, etoposide phosphate, and teniposide.

In some embodiments, the invention provides a method of treating cancerin an individual, comprising administering to the individual a) aneffective amount of a composition comprising nanoparticles comprising arapamycin or a derivative thereof and a carrier protein (e.g., albumin),and b) an effective amount of an antiangiogenic agent. In someembodiments, the invention provides a method of treating cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising rapamycinand an albumin, and b) an effective amount of an antiangiogenic agent.In some embodiments, the method is for treatment of metastatic breastcancer, breast cancer in an adjuvant setting or a neoadjuvant setting,lung cancer (such as first line advanced NSCLC and NSCLC), ovariancancer, and melanoma (including metastatic melanoma).

Many anti-angiogenic agents have been identified and are known in theart, including those listed by Carmeliet and Jain (2000). Theanti-angiogenic agent can be naturally occurring or non-naturallyoccurring. In some embodiments, the chemotherapeutic agent is asynthetic antiangiogenic peptide. For example, it has been previouslyreported that the antiangiogenic activity of small synthetic pro-apopticpeptides comprise two functional domains, one targeting the CD13receptors (aminopeptidase N) on tumor microvessels and the otherdisrupting the mitochondrial membrane following internalization. Nat.Med. 1999, 5(9):1032-8. A second generation dimeric peptide, CNGRC-GG-d(KLAKLAK)₂, named HKP (Hunter Killer Peptide) was found to have improvedantitumor activity. Accordingly, in some embodiments, the antiangiogenicpeptide is HKP. In some embodiments, the antiangiogenic agent is otherthan an anti-VEGF antibody (such as Avastin®). In some embodiments, theantiangiogenic agent is a small molecule inhibitor of VEGFR (such asVEGFR1, VEGFR2, and/or VEGFR3). Suitable small molecule inhibitors ofVEGFR include, but are not limited to, vatalanib, AZD2171, pazopanib(GW786034), Sunitinib, AG013736, Sorafenib, ZD6474, XL647, and XL999.

In some embodiments, the invention provides a method of treating cancerin an individual, comprising administering to the individual a) aneffective amount of a composition comprising nanoparticles comprising arapamycin or a derivative thereof and a carrier protein (e.g., albumin),and b) an effective amount of a proteasome inhibitor, such as bortezomib(Velcade). In some embodiments, the invention provides a method oftreating cancer in an individual, comprising administering to theindividual a) an effective amount of a composition comprisingnanoparticles comprising rapamycin and an albumin, and b) an effectiveamount of a proteasome inhibitor such as bortezomib (Velcade).

In some embodiments, the invention provides a method of treating cancerin an individual, comprising administering to the individual a) aneffective amount of a composition comprising nanoparticles comprising arapamycin or a derivative thereof and a carrier protein. (e.g.,albumin), and b) an effective amount of a therapeutic antibody. In someembodiments, the invention provides a method of treating cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising rapamycinand an albumin, and b) an effective amount of a therapeutic antibody.Suitable therapeutic antibodies include, but are not limited to,anti-VEGF antibody (such as Avastin® (bevacizumab)), anti-HER2 antibody(such as Herceptin® (trastuzumab)), Erbitux® (cetuximab), Campath(alemtuzumab), Myelotarg (gemtuzumab), Zevalin (ibritumomab tiuextan,Rituxan (rituximab), and Bexxar (tositumomab)). In some embodiments, thechemotherapeutic agent is Erbitux® (cetuximab). In some embodiments, thechemotherapeutic agent is a therapeutic antibody other than an antibodyagainst VEGF or HER2. In some embodiments, the method is for treatmentof HER2 positive breast cancer, including treatment of advanced breastcancer, treatment of metastatic cancer, treatment of breast cancer in anadjuvant setting, and treatment of cancer in a neoadjuvant setting. Insome embodiments, the method is for treatment of any of metastaticbreast cancer, breast cancer in an adjuvant setting or a neoadjuvantsetting, lung cancer (such as first line advanced NSCLC and NSCLC),ovarian cancer, head and neck cancer, and melanoma (including metastaticmelanoma). For example, in some embodiments, there is provided a methodfor treatment of HER2 positive metastatic breast cancer in anindividual, comprising administering to the individual about 54 mg to540 mg rapamycin or about 30 mg/m2 to 300 mg/m2 rapamycin in ananoparticle composition weekly for three weeks with the fourth weekoff, concurrent with the administration of Herceptin®.

In some embodiments, there is provided a method of treating cancer in anindividual, comprising administering to the individual: a) an effectiveamount of a composition comprising nanoparticles comprising rapamycin ora derivative thereof and a carrier protein (e.g., albumin), and b) aneffective amount of an anti-VEGF antibody. In some embodiments, theeffective amounts of the rapamycin or a derivative thereof nanoparticlecomposition and the anti-VEGF antibody synergistically inhibit cellproliferation (such as tumor cell growth). In some embodiments, at leastabout 10% (including for example at least about any of about 20%, 30%,40%, 60%, 70%, 80%, 90%, or 100%) cell proliferation is inhibited. Insome embodiments, the rapamycin or a derivative thereof is rapamycin. Insome embodiments, the anti-VEGF antibody is bevacizumab (such asAvastin®). In some embodiments, the rapamycin or a derivative thereof inthe nanoparticle in the composition is administered by intravenousadministration. In some embodiments, the anti-VEGF antibody isadministered by intravenous administration. In some embodiments, boththe rapamycin or a derivative thereof in the nanoparticle compositionand the anti-VEGF antibody are administered by intravenousadministration.

In some embodiments, there is provided a method of inhibiting tumormetastasis in an individual, comprising administering to the individual:a) an effective amount of a composition comprising nanoparticlescomprising rapamycin or a derivative thereof and a carrier protein(e.g., albumin), and b) an effective amount of an anti-VEGF antibody. Insome embodiments, the effective amounts of the rapamycin or a derivativethereof nanoparticle composition and the anti-VEGF antibodysynergistically inhibit tumor metastasis. In some embodiments, at leastabout 10% (including for example at least about any of about 20%, 30%,40%, 60%, 70%, 80%, 90%, or 100%) metastasis is inhibited. In someembodiments, method of inhibiting metastasis to lymph node is provided.In some embodiments, method of inhibiting metastasis to the lung isprovided. In some embodiments, the rapamycin or a derivative thereof israpamycin. In some embodiments, the anti-VEGF antibody is bevacizumab(such as Avastin®). In some embodiments, the rapamycin or a derivativethereof in the nanoparticle in the composition is administered byintravenous administration. In some embodiments, the anti-VEGF antibodyis administered by intravenous administration. In some embodiments, boththe rapamycin or a derivative thereof in the nanoparticle compositionand the anti-VEGF antibody are administered by intravenousadministration.

In some embodiments, two or more chemotherapeutic agents areadministered in addition to the rapamycin or a derivative thereof in thenanoparticle composition. These two or more chemotherapeutic agents may(but not necessarily) belong to different classes of chemotherapeuticagents. Examples of these combinations are provided herein. Othercombinations are also contemplated.

In some embodiments, there is provided a method of treating cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising a rapamycinor a derivative thereof and a carrier protein (e.g., albumin), b) aneffective amount of an antimetabolite (such as a nucleoside analog, forexample, gemcitabine), and c) an anthracycline antibiotic (such asepirubicin). In some embodiments, there is provided a method of treatingcancer in an individual, comprising administering to the individual a)an effective amount of a composition comprising nanoparticles comprisingrapamycin and an albumin, b) an effective amount of an antimetabolite(such as a nucleoside analog, for example, gemcitabine), and c) aneffective amount of an anthracycline antibiotic (such as epirubicin). Insome embodiments, the method is for treatment of breast cancer in aneoadjuvant setting. For example, in some embodiments, there is provideda method of treating locally advanced/inflammatory cancer in anindividual comprising administering to the individual rapamycin (such asabout 30 mg/m2 to about 300 mg/m2 or such as about 50 mg to 540 mgrapamycin) in a nanoparticle composition every two weeks; 2000 mg/m2gemcitabine, every two weeks; and 50 mg/m2 epirubicin, every two weeks.In some embodiments, there is provided a method of treating breastcancer in an individual in an adjuvant setting, comprising administeringto the individual rapamycin (such as about 30 mg/m2 to about 300 mg/m2or such as about 50 mg to 540 mg rapamycin) in a nanoparticlecomposition every two weeks, 2000 mg/m2 gemcitabine, every two weeks,and 50 mg/m2 epirubicin, every two weeks.

In some embodiments, there is provided a method of treating cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising a rapamycinor a derivative thereof and a carrier protein (e.g., albumin), b) aneffective amount of a platinum-based agent (such as carboplatin), and c)a therapeutic antibody (such as ant-HER2 antibody (such as Herceptin®)and anti-VEGF antibody (such as Avastin®)). In some embodiments, thereis provided a method of treating cancer in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising rapamycin and an albumin, b) aneffective amount of a platinum-based agent (such as carboplatin), and c)a therapeutic antibody (such as ant-HER2 antibody (such as Herceptin®)and anti-VEGF antibody (such as Avastin®)). In some embodiments, themethod is for treatment of any of advanced breast cancer, metastaticbreast cancer, breast cancer in an adjuvant setting, and lung cancer(including NSCLC and advanced NSCLC). In some embodiments, there isprovided a method of treating metastatic cancer in an individual,comprising administering to the individual rapamycin (such as about 30mg/m2 to about 300 mg/m2 or such as about 50 mg to 540 mg rapamycin) ina nanoparticle composition and carboplatin, AUC=2, wherein theadministration is carried out weekly for three weeks with the fourthweek off. In some embodiments, the method further comprises weeklyadministering about 2-4 Mg/kg of Herception®.

In some embodiments, there is provided a method of treating cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising a rapamycinor a derivative thereof and a carrier protein (e.g., albumin), b) aneffective amount of a platinum-based agent (such as carboplatin), and c)a vinca alkaloid (such as Navelbine®). In some embodiments, there isprovided a method of treating cancer in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising rapamycin and an albumin, b) aneffective amount of a platinum-based agent (such as carboplatin), and c)a vinca alkaloid (such as Navelbine®). In some embodiments, the methodis for treatment of lung cancer.

In some embodiments, the invention provides a method of treating cancerin an individual, comprising administering to the individual a) aneffective amount of a composition comprising nanoparticles comprising arapamycin or a derivative thereof and a carrier protein (e.g., albumin),b) an effective amount of an alkylating agent (such as cyclophosphamide)and c) an anthracycline antibiotic (such as adriamycin). In someembodiments, the invention provides a method of treating cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising rapamycinand an albumin, b) an effective amount of an alkylating agent (such ascyclophosphamide) and c) an anthracycline antibiotic (such asadriamycin). In some embodiments, the method is for treatment of anearly stage breast cancer. In some embodiments, the method is fortreatment of a breast cancer in an adjuvant or a neoadjuvant setting.For example, in some embodiments, there is provided a method of treatingan early stage breast cancer in an individual, comprising administeringrapamycin (such as about 30 mg/m2 to about 300 mg/m2 or 50 mg to 540 mgrapamycin) in a nanoparticle composition, 60 mg/m2 adriamycin, and 600mg/m2 cyclophosphamide, wherein the administration is carried out onceevery two weeks.

In some embodiments, the invention provides a method of treating cancerin an individual, comprising administering to the individual a) aneffective amount of a composition comprising nanoparticles comprising arapamycin or a derivative thereof and a carrier protein (e.g., albumin)and b) an effective amount of an p110α-specific inhibitor (e.g.,PX-866). In some embodiments, the method further comprises administeringan effective amount of a tyrosine kinase inhibitor (e.g., gefitinib orerlotinib). In some embodiments, the cancer is non-small cell lungcarcinoma.

In some embodiments, the invention provides a method of treating cancerin an individual, comprising administering to the individual a) aneffective amount of a composition comprising nanoparticles comprising arapamycin or a derivative thereof and a carrier protein (e.g., albumin)and b) an effective amount of a compound that affects the MAPK pathway(e.g., sorafenib (BAY49-9006). In some embodiments, the method furthercomprises administering an effective amount of a tyrosine kinaseinhibitor (e.g., gefitinib or erlotinib). In some embodiments, thecancer is non-small cell lung carcinoma. In some embodiments, the canceris brain cancer (e.g., glioblastoma).

In some embodiments, there is provided a method of treating cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising a rapamycinor a derivative thereof and a carrier protein (e.g., albumin) and b) aneffective amount of another agent that affects a signaling pathwayinvolving a target of rapamycin. In some embodiments, there is provideda method of treating cancer in an individual, comprising administeringto the individual a) an effective amount of a composition comprisingnanoparticles comprising a rapamycin or a derivative thereof and acarrier protein (e.g., albumin) and b) an effective amount of anotheragent that affects a signaling pathway involving mTOR. In someembodiments, the other agent affects a signaling pathway involvingTORC1. In some embodiments, the other agent affects a signaling pathwayinvolving mTORC2. Signaling pathways involving mTOR include, but are notlimited to, PI3K/Akt pathwayn and cAMP/AMPK pathway. These pathways areinterrelated. Accordingly, an agent that affects one signaling pathwayfrequently affects the other pathway (either directly or indirectly).

In some embodiments, the signaling pathway involving mTOR is thePI3K/Akt signaling pathway. For example, in some embodiments, there isprovided a method of treating cancer in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising a rapamycin or a derivative thereofand a carrier protein (e.g., albumin) and b) an effective amount ofanother agent that inhibits PI3K/Akt activation. In some embodiments,the cancer is any of HER2+ breast cancer, chronic mylogenous leukemiaCML, ovarian cancer, endometrial cancer, sarcoma, SCCHN (squamous cellcarcinomaterm of the head and neck), and thyroid cancer.

The PI3/Akt signaling pathway described herein includes any members orcomponents that directly or indirectly participate in the signaltransduction cascade. These include, but are not limited to, PI3 kinase,Akt, PDK1, RAPTOR (regulatory associated protein if mTOR), TSC1(tuberous sclerosis complex 1), TSC2, PTEN (phosphatase and tenesinhomolog), and downstream effectors such as cyclin D, HIF1, HIF2, Glut1,LAT1, and c-Myc. Components of the PI3/Akt signaling pathway may alsoinclude RHEB, Rictor, S6K, 4EBP1, cAMP, cAMPK, GβL, IRS, PIP2, PIP3,Rho, Ras, Abl, PKC, eIF4E, PDGFR, VEGFR, and VHL. The agent that affects(such as inhibits) the PI3K/Akt signaling pathway can thus act throughmodulation of any one or more of these components.

In some embodiments, the other agent inhibits PI3 kinase (PI3K).Suitable inhibitors of PI3K include, but are not limited to, wortmanninand the derivatives or analogs thereof; celecoxib and analogs thereof,such as OSU-03012 and OSU-03013; 3-deoxy-D-myo-inositol analogs, such asPX-316; 2′-substituted 3′-deoxy-phosphatidyl-myo-inositol analogs; fusedheteroaryl derivatives; 3-(imidazo[1,2-a]pyridin-3-yl) derivatives;Ly294002; quinazoline-4-one derivatives, such as IC486068;3-(hetero)aryloxy substituted benzo(b)thiophene derivatives; viridins,including semi-synthetic viridins such as such as PX-866 (acetic acid(1S,4E,10R,11R,13S,14R)-[4-diallylaminomethylene-6-hydroxy-1-methoxymethyl-10,13-dimethyl-3,7,17-trioxo-1,3,4,7,10,11,12,13,14,15,16,17-dodecahydro-2-oxa-cyclopenta[a]phenanthren-1′-ylester); and wortmannin and derivatives thereof.

In some embodiments, the other agent inhibits Akt kinase, includingAkt1, Akt2, and Akt3. In some embodiments, the other agent inhibitsphosphorylation of 5473 of the human Akt kinase, but not T308. In someembodiments, the second compound inhibits phosphorylation of T308 of thehuman Akt kinase, but not S473. In some embodiments, the other agentinhibits phorphorylation of both S473 and T308 of the Akt kinase. Insome embodiments, the other agent interferes with the membranelocalization of the Akt kinase. Suitable inhibitors of Akt kinaseinclude, but are not limited to, Akt-1-1 (inhibits Akt1), Akt-1-1,2(inhibits Akt1 and 2), API-59CJ-Ome, 1-H-imidazo[4,5-c]pyridinylcompounds, indole-3-carbinol and derivatives thereof, perifosine,phosphatidylinositol ether lipid analogues, triciribine (TCN or API-2 orNCI identifier: NSC 154020). In some embodiments, the other agent isperifosine.

In some embodiments, the other agent is an inhibitor of PDK1.

In some embodiments, there is provided a method of treating cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising a rapamycinor a derivative thereof and a carrier protein (e.g., albumin) and b) aneffective amount of another agent that inhibits cyclin D1 (such ascycline D1 overexpression). In some embodiments, the cancer is any ofmantle cell lymphoma and breast cancer.

In some embodiments, there is provided a method of treating cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising a rapamycinor a derivative thereof and a carrier protein (e.g., albumin) and b) aneffective amount of another agent that inhibits Myc over expression. Insome embodiments, the cancer is burkitt lymphoma.

In some embodiments, the other agent inhibits HIF. In some embodiments,the HIF is HIF 1. In some embodiments, the HIF is HIF2. In someembodiments, there is provided a method of treating cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising a rapamycinor a derivative thereof and a carrier protein (e.g., albumin) and b) aneffective amount of another agent that inhibits HIF (such as HIFoverexpression). In some embodiments, the other agent inhibitsHIF-mediated angiogenesis. In some embodiments, the cancer is RCC andVon Hippel-Lindau (VHL).

Other PI3KIAkt signaling pathway inhibitors include, but are not limitedto, e.g., FTY720 and UCN-01.

While the agents described herein are sometimes referred to as signalingpathway inhibitors, the methods described herein includes the use ofthese inhibitors to treat cancer regardless of the mechanism of actionor how the therapeutic effect is achieved. Indeed, it is recognized thatsuch compounds may have more than one target, and the initial activityrecognized for a compound may not be the activity that it possesses invivo when administered to a subject, or whereby it achieves itstherapeutic efficacy. Thus, the description of a compound as a pathwayor protein target (e.g., Akt or mTOR) inhibitor indicates that acompound possesses such activity, but in no way restricts a compound tohaving that activity when used as a therapeutic or prophylactic agent.

Other agents that can be used in combination with rapamycin (or itsderivative) compositions described herein include, for example,flavopiridol, antifolates, SN38, inhibitor of breast cancer resistantprotein (such as KO143 and fumitremorgin C).

In some embodiments, there is provided a method of treating advancedbreast cancer in an individual, comprising administering to theindividual a) an effective amount of a composition comprisingnanoparticles comprising a rapamycin and an albumin, b) an effectiveamount of carboplatin. In some embodiments, the method further comprisesadministering an effective amount of Herceptin® to the individual. Insome embodiments, there is provided a method of treating metastaticbreast cancer in an individual, comprising administering to theindividual a) an effective amount of a composition comprisingnanoparticles comprising rapamycin and an albumin, b) an effectiveamount of gemcitabine. In some embodiments, there is provided a methodof treating advanced non-small cell lung cancer in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising rapamycin and analbumin, b) an effective amount of carboplatin.

In some embodiments, the method further comprises identifying a cancerpatient population (e.g., breast cancer) based on a hormone receptorstatus of patients having tumor tissue not expressing both ER and PgRand administering to the patient population an effective amount of acomposition comprising nanoparticles comprising rapamycin or aderivative thereof and a carrier protein (e.g., albumin). In someembodiments, the method further comprises administering to the patientpopulation an effective amount of at least one other chemotherapeuticagent. The at least one other chemotherapeutic agent may be administeredconcurrently or sequentially with rapamycin or a derivative thereofnanoparticles. In some embodiments, the at least one otherchemotherapeutic agent comprises 5-Fluoruracil, Epirubicin andCyclophosphamide (FEC) administered concurrently or sequentially. Thesemethods may have higher efficacy in ER(−)/PgR(−) populations in allpatient populations, both HER-2 positive and HER-2 negative.

In some embodiments of any of the above methods of combination therapywith a chemotherapeutic agent, there is provided a compositioncomprising nanoparticles comprising rapamycin or a derivative thereofand a carrier protein (such as albumin) and at least one otherchemotherapeutic agent. The compositions described herein may compriseeffective amounts of the rapamycin or a derivative thereof and thechemotherapeutic agent for the treatment of a cancer. In someembodiments, the chemotherapeutic agent and rapamycin or a derivativethereof are present in the composition at a predetermined ratio, such asthe weight ratios described herein. In some embodiments, the inventionprovides a synergistic composition of an effective amount of acomposition comprising nanoparticles comprising rapamycin or aderivative thereof and an effective amount of at least one otherchemotherapeutic agent.

In some embodiments of any of the above methods of combination therapywith a chemotherapeutic agent, the invention provides pharmaceuticalcompositions comprising nanoparticles comprising a rapamycin or aderivative thereof and a carrier protein (such as albumin) for use inthe treatment of a cancer, wherein said use comprises simultaneousand/or sequential administration of at least one other chemotherapeuticagent. In some embodiments, the invention provides a pharmaceuticalcomposition comprising a chemotherapeutic agent for use in the treatmentof a cancer, wherein said use comprises simultaneous and/or sequentialadministration of a composition comprising nanoparticles comprisingrapamycin or a derivative thereof and a carrier protein (such asalbumin). In some embodiments, the invention provides rapamycin or aderivative thereof-containing nanoparticle compositions and compositionscomprising one other chemotherapeutic agent for simultaneous, and/orsequential use for treatment of a cancer.

In some embodiments, the invention provides a method to treat cancercomprising administering to an individual an effective amount of acomposition comprising nanoparticles comprising a rapamycin or aderivative thereof and a carrier protein (such as albumin)simultaneously and/or sequentially with surgery, radiation, genetherapy, immunotherapy, bone marrow transplantation, stem celltransplantation, hormone therapy, targeted therapy, cryotherapy,ultrasound therapy, and/or photodynamic therapy. In some embodiments,the present invention provides a method of treating cancer comprising afirst therapy comprising administering nanoparticles comprisingrapamycin and an albumin, and a second therapy comprising surgery,radiation, gene therapy, immunotherapy, bone marrow transplantation,stem cell transplantation, hormone therapy, targeted therapy,cryotherapy, ultrasound therapy, and/or photodynamic therapy. In someembodiments, the cancer may be prostate cancer. In some embodiments, thesecond therapy is hormone therapy. In some embodiments, the secondtherapy is radiation therapy. In some embodiments, the second therapy issurgery.

The administration of rapamycin or a derivative thereof nanoparticlecomposition may be prior to the hormone therapy, radiation, and/orsurgery, after the hormone therapy, radiation, and/or surgery, orconcurrent with hormone therapy, radiation, and/or surgery. For example,the administration of rapamycin or a derivative thereof nanoparticlecomposition may precede or follow hormone therapy, radiation, and/orsurgery therapy by intervals ranging from minutes to weeks. In someembodiments, the time period between the first and the second therapy issuch that the rapamycin or a derivative thereof and a carrier protein(e.g., albumin) and hormone therapy, radiation, and/or surgery wouldstill be able to exert an advantageously combined effect on the cell. Insome embodiments, it may be desirable to extend the time period fortreatment significantly, where several days to several weeks lapsebetween the two therapies.

Surgery described herein includes resection in which all or part ofcancerous tissue is physically removed, exercised, and/or destroyed.Tumor resection refers to physical removal of at least part of a tumor.In addition to tumor resection, treatment by surgery includes lasersurgery, cryosurgery, electrosurgery, and micropically controlledsurgery (Mohs surgery). Removal of superficial surgery, precancers, ornormal tissues are also contemplated.

The hormone therapy, radiation therapy, and/or surgery may be carriedout in addition to the administration of chemotherapeutic agents. Forexample, the individual may first be administered with a rapamycin orits derivative thereof-containing nanoparticle composition and at leastone other chemotherapeutic agent, and subsequently be subject to hormonetherapy, radiation therapy, and/or surgery. Alternatively, theindividual may first be treated with hormone therapy, radiation therapy,and/or surgery, which is then followed by the administration of ananoparticle composition and at least one other chemotherapeutic agent.Other combinations are also contemplated.

Administration of nanoparticle compositions disclosed above inconjunction with administration of chemotherapeutic agent is equallyapplicable to those in conjunction with hormone therapy, radiationtherapy, and/or surgery.

The term hormone therapy, as used herein, includes, but is not limitedto, androgen ablation therapy, androgen deprivation therapy, hormonalablation therapy, combined hormone blockade, intermittent hormonaltherapy, neoadjuvant hormonal therapy, neoadjuvant androgen suppression,and neoadjuvant androgen deprivation. Androgens, such as testosterone,regulate the growth, differentiation, and rate of apoptosis in theprostate and its malignancies. In some embodiments, prostate cancer maybe treated by exploiting the general dependency of prostate cancer onandrogen through several therapies referred to as hormone therapy.

In some embodiments, there is provided a method of treating cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising a rapamycinor a derivative thereof and a carrier protein (e.g., albumin) and b) aneffective amount of a gonadotropin-releasing hormone (GnRH) agonist(also called LHRH agonist, luteinizing-hormone releasing hormoneagonist). In some embodiments, there is provided a method of treatingcancer in an individual, comprising administering to the individual a)an effective amount of a composition comprising nanoparticles comprisingrapamycin and an albumin and b) an effective amount of a GnRH agonist.In some embodiments, the method is for treatment of prostate cancer. Insome embodiments, the invention provides pharmaceutical compositionscomprising nanoparticles comprising a rapamycin or a derivative thereofand a carrier protein (such as albumin) for use in the treatment of acancer, wherein said use comprises simultaneous and/or sequentialadministration of at least one GnRH agonist. Suitable therapeutic GnRHagonists include, but are not limited to, leuprolide, goserelin,naferelin, meterelin, buserelin, historelin, deslorelin, andtriptorelin.

In some embodiments, there is provided a method of treating cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising a rapamycinor a derivative thereof and a carrier protein (e.g., albumin), b) aneffective amount of a GnRH agonist, and c) antiandrogen. In someembodiments, there is provided a method of treating cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising rapamycinand an albumin, b) an effective amount of a GnRH agonist, c) and anantiandrogen. In some embodiments, the method is for treatment ofprostate cancer. In some embodiments, the antiandrogen administrationbegins prior to treatment with the GnRH agonist and/or therapamycin-containing nanoparticle composition. In some embodiments, theinvention provides pharmaceutical compositions comprising nanoparticlescomprising a rapamycin or a derivative thereof and a carrier protein(such as albumin) for use in the treatment of a cancer, wherein said usecomprises simultaneous and/or sequential administration of at least oneGnRH agonist or antiandrogen. In some embodiments, the antiandrogen isadministered before the GnRH agonist and/or the rapamycin-containingnanoparticle composition, and the administration of the antiandrogen iscontinued for at least the first month of GnRH agonist therapy. In someembodiments, the antiandrogen administration begins any of about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, and 24 weeks prior to treatment with the GnRH agonist and/or therapamycin-containing nanoparticle composition Suitable therapeutic GnRHagonists include, but are not limited to, leuprolide, goserelin,naferelin, meterelin, buserelin, historelin, deslorelin, andtriptorelin. Suitable therapeutic antiandrogens include, but are notlimited to, bicalutamide (Casodex), flutamide (Eulexin), cyproterone,nilutamide (Nilandron), and other therapeutic agents that are effectivein ultimately reducing circulating androgen levels to the castrate level

In some embodiments, there is provided a method of treating cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising a rapamycinor a derivative thereof and a carrier protein (e.g., albumin) and b) aneffective amount of a gonadotropin-releasing hormone (GnRH) antagonist(also called LHRH antagonist, luteinizing-hormone releasing hormoneantagonist). In some embodiments, there is provided a method of treatingcancer in an individual, comprising administering to the individual a)an effective amount of a composition comprising nanoparticles comprisingrapamycin and an albumin and b) an effective amount of a GnRHantagonist. In some embodiments, the method is for treatment of prostatecancer. In some embodiments, the invention provides pharmaceuticalcompositions comprising nanoparticles comprising a rapamycin or aderivative thereof and a carrier protein (such as albumin) for use inthe treatment of a cancer, wherein said use comprises simultaneousand/or sequential administration of at least one GnRH antagonist.Suitable therapeutic GnRH antagonist include, but are not limited to,Cetrorelix acetate (Cetrotide), by Serono, Ganirelix acetate (Antagon),by Organon International, Abarelix (Plenaxis), and the like.

In an embodiment, the method comprises the administration of atherapeutic effective amount of a rapamycin-containing nanoparticlecomposition at any one or more of the following times: prior to hormonetherapy, in conjunction with hormone therapy, during hormone therapy, orfollowing hormone therapy to treat prostate cancer. In some embodiments,the method comprises the administration of a therapeutic effectiveamount of a rapamycin-containing nanoparticle composition eithersimultaneously with or separately from the hormone therapeutic agent totreat prostate cancer. A combination of a therapeutically effectiveamount of one or more standard hormone therapy drugs and atherapeutically effective amount of rapamycin or a derivative in ananoparticle composition may result in a synergistic effect in prostatetumor inhibition (including regression of existing prostate tumor).

In some embodiments, there is provided a method of treating cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising a rapamycinor a derivative thereof and a carrier protein (e.g., albumin) and b) aneffective amount of an endothelin-A receptor blocker, inhibitor, orantagonist. In some embodiments, there is provided a method of treatingcancer in an individual, comprising administering to the individual a)an effective amount of a composition comprising nanoparticles comprisingrapamycin and an albumin and b) an effective amount of an endothelin-Areceptor blocker, inhibitor, or antagonist. In some embodiments, themethod is for treatment of prostate cancer (such as advanced prostatecancer). In some embodiments, the invention provides pharmaceuticalcompositions comprising nanoparticles comprising a rapamycin or aderivative thereof and a carrier protein (such as albumin) for use inthe treatment of a cancer, wherein said use comprises simultaneousand/or sequential administration of at least one endothelin-A receptorblocker, inhibitor, or antagonist. A suitable therapeutic endothelin-Areceptor blocker, inhibitor, or antagonist includes, but is not limitedto, Atrasentan (ABT 627, Abbott Laboratories, Abbott Park, Ill.).

It is understood that any of the methods of treating cancer describedherein (such as above section “Methods of Treating Cancer”) apply to andinclude description of combination therapies. In some embodiments of anyof the methods of treatment related to combination therapy describedherein, treatment with the combination of the first therapy (e.g., ananoparticle composition comprising rapamycin or a derivative thereofand a carrier protein) and the second therapy (e.g., one or morecompounds useful for treating cancer) may result in an additive or evensynergistic (e.g., greater than additive) result compared toadministration of either therapy alone. In some embodiments, a loweramount of each pharmaceutically active compound is used as part of acombination therapy compared to the amount generally used for individualtherapy. Preferably, the same or greater therapeutic benefit is achievedusing a combination therapy than by using any of the individualcompounds alone. In some embodiments, the same or greater therapeuticbenefit is achieved using a smaller amount (e.g., a lower dose or a lessfrequent dosing schedule) of a pharmaceutically active compound in acombination therapy than the amount generally used for individualtherapy. Preferably, the use of a small amount of pharmaceuticallyactive compound results in a reduction in the number, severity,frequency, or duration of one or more side-effects associated with thecompound.

In some embodiments of any of the methods of treatment related tocombination therapy, the rapamycin or derivative thereof and the secondcompound (e.g., a chemotherapeutic agent and/or hormone therapeuticagent) are present in a single composition containing at least twodifferent nanoparticles, wherein some of the nanoparticles in thecomposition comprise the rapamycin or derivative thereof and a carrierprotein, and some of the other nanoparticles in the composition comprisea second pharmaceutically active compound and a carrier protein. In someembodiments, only the rapamycin or derivative thereof is contained innanoparticles. In some embodiments, simultaneous administration ofrapamycin or derivative thereof in the nanoparticle composition and thesecond compound can be combined with supplemental doses of rapamycinand/or the second compound.

In some embodiments of any of the above embodiments related tocombination therapies described herein, the first and second therapiesare administered simultaneously, either in the same composition or inseparate compositions. In some embodiments, the first and secondtherapies are administered sequentially, i.e., the first therapy isadministered either prior to or after the administration of the firstand second therapy. In some embodiments, the administration of the firstand second therapies is concurrent, i.e., the administration period ofthe first therapy and that of the second therapy overlap with eachother. In some embodiments, the administration of the first and secondtherapies is non-concurrent. For example, in some embodiments, theadministration of the first therapy is terminated before the secondtherapy is administered. In some embodiments, the administration of thesecond therapy is terminated before the first therapy is administered.In some embodiments, the second therapy is radiation therapy. In someembodiments, the second therapy is surgery.

In some embodiments of any of the above embodiments related tocombination therapy, the cancer is early stage cancer, non-metastaticcancer, primary cancer, advanced cancer, locally advanced cancer,metastatic cancer, cancer in remission, recurrent cancer, cancer in anadjuvant setting, cancer in a neoadjuvant setting, or cancersubstantially refractory to hormone therapy. In some embodiments, thecancer is a solid tumor. In some embodiments, the cancer is not a solidtumor (i.e., other than a solid tumor). In some embodiments, the canceris a plasmacytoma. In some embodiments, the cancer is multiple myeloma,renal cell carcinoma, prostate cancer, lung cancer, melanoma, braincancer (e.g., glioblastoma), ovarian cancer, or breast cancer. In someembodiments, the cancer is a carcinoma (i.e., other than a carcinoma).In some embodiments, the cancer is not colon cancer (i.e., other thancolon cancer). In some embodiments, the cancer is not breast cancer(i.e., other than breast cancer). In some embodiments, the cancer is notovarian cancer, prostate cancer, or brain cancer.

In some embodiments of any of the above embodiments related tocombination therapy, a taxane is not administered to the individual. Insome embodiments, the taxane administered is not a nanoparticlecomposition. In some embodiments, the nanoparticle compositioncomprising rapamycin or a derivative thereof is not administered inconjunction with a taxane. In some embodiments, a taxane is notadministered to the individual during the time period in which theindividual is receiving one or more doses of a nanoparticle compositioncomprising rapamycin or a derivative thereof. In some embodiments, theindividual was treated with a taxane before treatment begins with ananoparticle composition comprising rapamycin or a derivative thereof.For example, the individual may have received a taxane one or more days,weeks, months, or years before treatment begins with a nanoparticlecomposition comprising rapamycin or a derivative thereof. In otherembodiments, the individual never receives a taxane before treatmentbegins with a nanoparticle composition comprising rapamycin or aderivative thereof. In some embodiments, the individual is treated witha taxane after treatment with a nanoparticle composition comprisingrapamycin or derivative thereof terminates. In other embodiments, theindividual is never treated with a taxane after treatment with ananoparticle composition comprising rapamycin or derivative thereofterminates. In some embodiments, the composition, first therapy, and/orsecond therapy do not contain a taxane. In other embodiments, thecomposition, first therapy, and/or second therapy comprise a taxane.

Dosing and Method of Administration

The dose of the inventive composition administered to an individual(such as a human) may vary with the particular composition, the methodof administration, and the particular stage of cancer being treated. Theamount should be sufficient to produce a desirable response, such as atherapeutic or prophylactic response against cancer. In someembodiments, the amount of the composition is a therapeuticallyeffective amount. In some embodiments, that amount of the composition isa prophylactically effective amount. In some embodiments, the amount ofrapamycin or a derivative thereof in the composition is below the levelthat induces a toxicological effect (i.e., an effect above a clinicallyacceptable level of toxicity) or is at a level where a potential sideeffect can be controlled or tolerated when the composition isadministered to the individual.

In some embodiments, the amount of rapamycin or a derivative thereof inthe composition is an amount sufficient to increase basal AKT activity,increase AKT phosphorylation, increase PI3-kinase activity, increase thelength of activation of AKT (e.g., activation induced by exogenousIGF-1), inhibit serine phosphorylation of IRS-1, inhibit IRS-1degradation, inhibit or alter CXCR4 subcellular localization, inhibitVEGF secretion, decrease expression of cyclin D2, decrease expression ofsurvivin, inhibit IL-6-induced multiple myeloma cell growth, inhibitcancer cell proliferation, increase apoptosis, increase cell cyclearrest, increase cleavage of poly(ADPribose) polymerase, increasecleavage of caspase-8/caspase-9, alter or inhibit signaling in thephosphatidylinositol 3-kinase/AKT/mTOR and/or cyclin D1/retinoblastomapathways, inhibit angiogenesis, and/or inhibit osteoclast formation.

In some embodiments, the invention provides a method of treating cancerin an individual by administering to the individual (e.g., a human) aneffective amount of a composition comprising nanoparticles that compriserapamycin or a derivative thereof and a carrier protein (e.g., albuminsuch as human serum albumin). In some embodiments, the amount ofrapamycin or a derivative thereof in the composition is included in anyof the following ranges: about 0.5 to about 5 mg, about 5 to about 10mg, about 10 to about 15 mg, about 15 to about 20 mg, about 20 to about25 mg, about 20 to about 50 mg, about 25 to about 50 mg, about 50 toabout 75 mg, about 50 to about 100 mg, about 75 to about 100 mg, about100 to about 125 mg, about 125 to about 150 mg, about 150 to about 175mg, about 175 to about 200 mg, about 200 to about 225 mg, about 225 toabout 250 mg, about 250 to about 300 mg, about 300 to about 350 mg,about 350 to about 400 mg, about 400 to about 450 mg, or about 450 toabout 500 mg. In some embodiments, the amount of rapamycin or derivativethereof in the effective amount of the composition (e.g., a unit dosageform) is in the range of about 54 mg to about 540 mg, such as about 180mg to about 270 mg or about 216 mg. In some embodiments, theconcentration of the rapamycin in the composition is dilute (about 0.1mg/ml) or concentrated (about 100 mg/ml), including for example any ofabout 0.1 to about 50 mg/ml, about 0.1 to about 20 mg/ml, about 1 toabout 10 mg/ml, about 2 mg/ml to about 8 mg/ml, about 4 to about 6mg/ml, about 5 mg/ml. In some embodiments, the concentration of therapamycin is at least about any of 0.5 mg/ml, 1.3 mg/ml, 1.5 mg/ml, 2mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10mg/ml, 15 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, or 50 mg/ml.

Exemplary effective amounts of rapamycin or a derivative thereof in thenanoparticle composition include, but are not limited to, about any of25 mg/m², 30 mg/m², 50 mg/m², 60 mg/m², 75 mg/m², 80 mg/m², 90 mg/m²,100 mg/m², 120 mg/m², 160 mg/m², 175 mg/m², 180 mg/m², 200 mg/m², 210mg/m², 220 mg/m², 250 mg/m², 260 mg/m², 300 mg/m², 350 mg/m², 400 mg/m²,500 mg/m², 540 mg/m², 750 mg/m², 1000 mg/m², or 1080 mg/m² rapamycin. Invarious embodiments, the composition includes less than about any of 350mg/m², 300 mg/m², 250 mg/m², 200 mg/m², 150 mg/m², 120 mg/m², 100 mg/m², 90 mg/m², 50 mg/m², or 30 mg/m² rapamycin or a derivative thereof.In some embodiments, the amount of the rapamycin or a derivative thereofper administration is less than about any of 25 mg/m², 22 mg/m², 20mg/m², 18 mg/m², 15 mg/m², 14 mg/m², 13 mg/m², 12 mg/m², 11 mg/m², 10mg/m², 9 mg/m², 8 mg/m², 7 mg/m², 6 mg/m², 5 mg/m², 4 mg/m², 3 mg/m², 2mg/m², or 1 mg/m². In some embodiments, the effective amount ofrapamycin or a derivative thereof in the composition is included in anyof the following ranges: about 1 to about 5 mg/m², about 5 to about 10mg/m², about 10 to about 25 mg/m², about 25 to about 50 mg/m², about 50to about 75 mg/m², about 75 to about 100 mg/m², about 100 to about 125mg/m², about 125 to about 150 mg/m², about 150 to about 175 mg/m², about175 to about 200 mg/m², about 200 to about 225 mg/m², about 225 to about250 mg/m², about 250 to about 300 mg/m², about 300 to about 350 mg/m²,or about 350 to about 400 mg/m². Preferably, the effective amount ofrapamycin or a derivative thereof in the composition is about 30 toabout 300 mg/m², such as about 100 to about 150 mg/m², about 120 mg/m²,about 130 mg/m², or about 140 mg/m².

In some embodiments of any of the above aspects, the effective amount ofrapamycin or a derivative thereof in the composition includes at leastabout any of 1 mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg,or 20 mg/kg. In various embodiments, the effective amount of rapamycinor a derivative thereof in the composition includes less than about anyof 350 mg/kg, 300 mg/kg, 250 mg/kg, 200 mg/kg, 150 mg/kg, 100 mg/kg, 50mg/kg, 25 mg/kg, 20 mg/kg, 10 mg/kg, 5 mg/kg, or 1 mg/kg rapamycin or aderivative thereof.

Exemplary dosing frequencies include, but are not limited to, weeklywithout break; weekly, three out of four weeks; once every three weeks;once every two weeks; weekly, two out of three weeks. In someembodiments, the composition is administered about once every 2 weeks,once every 3 weeks, once every 4 weeks, once every 6 weeks, or onceevery 8 weeks. In some embodiments, the composition is administered atleast about any of 1×, 2×, 3×, 4×, 5×, 6×, or 7× (i.e., daily) a week.In some embodiments, the intervals between each administration are lessthan about any of 6 months, 3 months, 1 month, 20 days, 15, days, 12days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2days, or 1 day. In some embodiments, the intervals between eachadministration are more than about any of 1 month, 2 months, 3 months, 4months, 5 months, 6 months, 8 months, or 12 months. In some embodiments,there is no break in the dosing schedule. In some embodiments, theinterval between each administration is no more than about a week.

The administration of the composition can be extended over an extendedperiod of time, such as from about a month up to about seven years. Insome embodiments, the composition is administered over a period of atleast about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36,48, 60, 72, or 84 months. In some embodiments, the rapamycin orderivative thereof is administered over a period of at least one month,wherein the interval between each administration is no more than about aweek, and wherein the dose of the rapamycin or a derivative thereof ateach administration is about 0.25 mg/m² to about 75 mg/m², such as about0.25 mg/m² to about 25 mg/m² or about 25 mg/m² to about 50 mg/m².

In some embodiments, the dosage of rapamycin in a nanoparticlecomposition can be in the range of 100-400 mg/m² when given on a 3 weekschedule, or 50-250 mg/m² when given on a weekly schedule. Preferably,the amount of rapamycin is about 80 to about 180 mg/m² (e.g., about 100mg/m² to about 150 mg/m², such as about 120 mg/m²).

Other exemplary dosing schedules for the administration of thenanoparticle composition (e.g., rapamycin/albumin nanoparticlecomposition) include, but are not limited to, 100 mg/m², weekly, withoutbreak; 75 mg/m² weekly, 3 out of four weeks; 100 mg/m², weekly, 3 out of4 weeks; 125 mg/m², weekly, 3 out of 4 weeks; 125 mg/m², weekly, 2 outof 3 weeks; 130 mg/m², weekly, without break; 175 mg/m², once every 2weeks; 260 mg/m², once every 2 weeks; 260 mg/m², once every 3 weeks;180-300 mg/m², every three weeks; 60-175 mg/m², weekly, without break;20-150 mg/m² twice a week; and 150-250 mg/m² twice a week. The dosingfrequency of the composition may be adjusted over the course of thetreatment based on the judgment of the administering physician.

In some embodiments, the composition is administered about 20 to about40 mg/kg three times weekly. In some embodiments, the composition isadministered about 60 to about 120 mg/m², three times weekly or about 90mg/m² daily. In some embodiments, the composition is administered about30 mg/kg daily. In some embodiments, methods of treating multiplemyeloma following these dosing regimes are provided.

In yet another aspect, the invention provides a method of treatingcancer in an individual by parenterally administering to the individual(e.g., a human) an effective amount of a composition comprisingnanoparticles that comprise rapamycin or a derivative thereof and acarrier protein (e.g., albumin such as human serum albumin). In someembodiments, the route of administration is intravenous, intra-arterial,intramuscular, or subcutaneous. In various embodiments, about 54 mg toabout 540 mg, such as about 180 mg to about 270 mg or about 216 mg, ofthe rapamycin or derivative thereof is administered per dose. In someembodiments, a taxane is not contained in the composition. In someembodiments, the rapamycin or derivative thereof is the onlypharmaceutically active agent for the treatment of cancer that iscontained in the composition.

The compositions described herein can be administered to an individual(such as human) via various routes, including, for example, intravenous,intra-arterial, intraperitoneal, intrapulmonary, oral, inhalation,intravesicular, intramuscular, intra-tracheal, subcutaneous,intraocular, intrathecal, transmucosal, and transdermal. In someembodiments, sustained continuous release formulation of the compositionmay be used. For example, the inventive composition can be administeredby inhalation to treat conditions of the respiratory tract. Thecomposition can be used to treat respiratory conditions such aspulmonary fibrosis, broncheolitis obliterans, lung cancer,bronchoalveolar carcinoma, and the like. In one embodiment of theinvention, nanoparticles (such as albumin nanoparticles) of theinventive compounds can be administered by any acceptable routeincluding, but not limited to, orally, intramuscularly, transdermally,intravenously, through an inhaler or other air borne delivery systemsand the like. In some embodiments, the rapamycin or derivative thereofis not coating a stent or is not administered using a stent.

The dosing frequency of the rapamycin-containing nanoparticlecomposition and the second compound may be adjusted over the course ofthe treatment based on the judgment of the administering physician. Insome embodiments, the first and second therapies are administeredsimultaneously, sequentially, or concurrently. When administeredseparately, the rapamycin-containing nanoparticle composition and thesecond compound can be administered at different dosing frequency orintervals. For example, the rapamycin-containing nanoparticlecomposition can be administered weekly, while a second compound can beadministered more or less frequently. In some embodiments, sustainedcontinuous release formulation of the rapamycin-containing nanoparticleand/or second compound may be used. Various formulations and devices forachieving sustained release are known in the art. A combination of theadministration configurations described herein can be used.

Modes of Administration of Combination Therapies

In some embodiments, the present invention provides a method of treatingcancer comprising a first therapy comprising administering nanoparticlescomprising rapamycin or a derivative thereof and a carrier protein(e.g., albumin) and a second therapy comprising chemotherapy and/orhormone therapy. In some embodiments, the method comprises a) a firsttherapy comprising administering to the individual a compositioncomprising nanoparticles of rapamycin and an albumin; and b) a secondtherapy comprising chemotherapy and/or hormone therapy.

The dose of the inventive composition administered to an individual(e.g., a human) in combination therapy may vary with the particularcomposition, the method of administration, and the particular stage ofcancer being treated. The amount should be sufficient to produce adesirable response, such as a therapeutic or prophylactic responseagainst cancer. In some embodiments, the amount of the composition is atherapeutically effective amount. In some embodiments, that amount ofthe composition is a prophylactically effective amount. In someembodiments, the amount of rapamycin or a derivative thereof in thecomposition is below the level that induces a toxicological effect(e.g., an effect above a clinically acceptable level of toxicity) or isat a level where a potential side effect can be controlled or toleratedwhen the composition is administered to the individual.

The composition comprising nanoparticles comprising rapamycin or aderivative thereof and a carrier protein (e.g., albumin) (also referredto as “nanoparticle composition”) and the chemotherapeutic agent and/orhormone therapeutic agent can be administered simultaneously (e.g.,simultaneous administration) and/or sequentially (e.g., sequentialadministration).

In some embodiments, the nanoparticle composition and thechemotherapeutic agent and/or hormone therapeutic agents (including thespecific chemotherapeutic agents described herein) are administeredsimultaneously. The term “simultaneous administration,” as used herein,means that the nanoparticle composition and the chemotherapeutic agentand/or hormone therapeutic agent are administered with a time separationof no more than about 15 minute(s), such as no more than about any of10, 5, or 1 minutes. When the drugs are administered simultaneously, therapamycin or a derivative thereof in the nanoparticles and thechemotherapeutic agent and/or hormone therapeutic agent may be containedin the same composition (e.g., a composition comprising both thenanoparticles and the chemotherapeutic agent) or in separatecompositions (e.g., the nanoparticles are contained in one compositionand the chemotherapeutic agent is contained in another composition). Forexample, rapamycin or a derivative thereof and a carrier protein (e.g.,albumin) and the chemotherapeutic agent may be present in a singlecomposition containing at least two different nanoparticles, whereinsome of the nanoparticles in the composition comprise rapamycin or aderivative thereof and a carrier protein, and some of the othernanoparticles in the composition comprise the chemotherapeutic agent anda carrier protein. The invention contemplates and encompasses suchcompositions. In some embodiments, only rapamycin or a derivativethereof is contained in nanoparticles. In some embodiments, simultaneousadministration of the rapamycin or a derivative thereof in thenanoparticle composition and the chemotherapeutic agent and/or hormonetherapeutic agent can be combined with supplemental doses of therapamycin or a derivative thereof and/or the chemotherapeutic agentand/or hormone therapeutic agent.

In some embodiments, the rapamycin or a derivative thereof nanoparticlecomposition and the chemotherapeutic agent and/or hormone therapeuticagent are administered sequentially. The term “sequentialadministration” as used herein means that the rapamycin or a derivativethereof in the nanoparticle composition and the chemotherapeutic agentand/or hormone therapeutic agent are administered with a time separationof more than about 15 minutes, such as more than about any of 20, 30,40, 50, 60 or more minutes. Either the rapamycin or a derivative thereofnanoparticle composition or the chemotherapeutic agent and/or hormonetherapeutic agent may be administered first. The rapamycin or aderivative thereof nanoparticle composition and the chemotherapeuticagent and/or hormone therapeutic agent are contained in separatecompositions, which may be contained in the same or different packages.

In some embodiments, the administration of the rapamycin or a derivativethereof nanoparticle composition and the chemotherapeutic agent and/orhormone therapeutic agent are concurrent, e.g., the administrationperiod of the nanoparticle composition and that of the chemotherapeuticagent and/or hormone therapeutic agent overlap with each other. In someembodiments, the administration of the rapamycin or a derivative thereofnanoparticle composition and the chemotherapeutic agent and/or hormonetherapeutic agent are non-concurrent. For example, in some embodiments,the administration of the rapamycin or a derivative thereof nanoparticlecomposition is terminated before the chemotherapeutic agent and/orhormone therapy is administered. In some embodiments, the administrationof the chemotherapeutic agent and/or hormone therapy is terminatedbefore the rapamycin or a derivative thereof nanoparticle composition isadministered. The time period between these two non-concurrentadministrations can range from about two to eight weeks, such as aboutfour weeks.

The dosing frequency of the rapamycin or a derivative thereof-containingnanoparticle composition and the chemotherapeutic agent and/or hormonetherapy may be adjusted over the course of the treatment, based on thejudgment of the administering physician. When administered separately,the rapamycin or a derivative thereof-containing nanoparticlecomposition and the chemotherapeutic agent and/or hormone therapy can beadministered at different dosing frequency or intervals. For example,the rapamycin or a derivative thereof-containing nanoparticlecomposition can be administered weekly, while a chemotherapeutic agentand/or hormone therapeutic agent can be administered more or lessfrequently. In some embodiments, sustained continuous releaseformulation of the rapamycin or a derivative thereof-containingnanoparticle and/or chemotherapeutic agent and/or hormone therapeuticagent may be used. Various formulations and devices for achievingsustained release are known in the art.

The rapamycin or a derivative thereof nanoparticle composition and thechemotherapeutic agent and/or hormone therapeutic agent can beadministered using the same route of administration or different routesof administration. In some embodiments (for both simultaneous andsequential administrations), the rapamycin or a derivative thereof inthe nanoparticle composition and the chemotherapeutic agent and/orhormone therapeutic agent are administered at a predetermined ratio. Forexample, in some embodiments, the ratio by weight of the rapamycin or aderivative thereof in the nanoparticle composition and thechemotherapeutic agent or the hormone therapeutic agent is about 1 to 1.In some embodiments, the weight ratio may be between about 0.001 toabout 1 and about 1000 to about 1, or between about 0.01 to about 1 and100 to about 1. In some embodiments, the ratio by weight of therapamycin or a derivative thereof in the nanoparticle composition andthe chemotherapeutic agent or hormone therapeutic agent is less than anyof about 100:1, 50:1, 30:1, 10:1, 9:1, 8:1, 7.5:1, 7:1, 6:1, 5:1, 4:1,3:1, 2:1, and 1:1. In some embodiments, the ratio by weight of therapamycin or a derivative thereof in the nanoparticle composition andthe chemotherapeutic agent or hormone therapeutic agent is more than anyof about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 7.5:1, 8:1, 9:1, 30:1, 50:1,100:1. Other ratios are contemplated.

The doses required for the rapamycin or a derivative thereof and/or thechemotherapeutic agent and/or hormone therapeutic agent may (but notnecessarily) be lower than what is normally required when each agent isadministered alone. Thus, in some embodiments, a subtherapeutic amountof the rapamycin or a derivative thereof in the nanoparticle compositionand/or the chemotherapeutic agent and/or hormone therapeutic agent areadministered. “Subtherapeutic amount” or “subtherapeutic level” refer toan amount that is less than the therapeutic amount, that is, less thanthe amount normally used when the rapamycin or a derivative thereof inthe nanoparticle composition and/or the chemotherapeutic agent and/orhormone therapeutic agent are administered alone. The reduction may bereflected in terms of the amount administered at a given administrationand/or the amount administered over a given period of time (reducedfrequency).

In some embodiments, enough chemotherapeutic agent and/or hormonetherapeutic agent is administered so as to allow reduction of the normaldose of the rapamycin or a derivative thereof in the nanoparticlecomposition required to effect the same degree of treatment by at leastabout any of 5%, 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or more. Insome embodiments, enough rapamycin or a derivative thereof in thenanoparticle composition is administered so as to allow reduction of thenormal dose of the chemotherapeutic agent and/or hormone therapeuticagent required to affect the same degree of treatment by at least aboutany of 5%, 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or more.

In some embodiments, the dose of both the rapamycin or a derivativethereof in the nanoparticle composition and the chemotherapeutic agentand/or hormone therapeutic agent are reduced as compared to thecorresponding normal dose of each when administered alone. In someembodiments, both the rapamycin or a derivative thereof in thenanoparticle composition and the chemotherapeutic agent and/or hormonetherapeutic agent are administered at a subtherapeutic, e.g., reducedlevel. In some embodiments, the dose of the nanoparticle compositionand/or the chemotherapeutic agent is substantially less than theestablished maximum toxic dose (MTD). For example, the dose of therapamycin or a derivative thereof nanoparticle composition and/or thechemotherapeutic agent and/or hormone therapeutic agent is less thanabout 50%, 40%, 30%, 20%, or 10% of the MTD.

A combination of the administration configurations described herein canbe used. The combination therapy methods described herein may beperformed alone or in conjunction with another therapy, such as surgery,radiation, gene therapy, immunotherapy, bone marrow transplantation,stem cell transplantation, hormone therapy, targeted therapy,cryotherapy, ultrasound therapy, photodynamic therapy, and/orchemotherapy and the like. Additionally, a person having a greater riskof developing the proliferative disease may receive treatments toinhibit or and/or delay the development of the disease.

As will be understood by those of ordinary skill in the art, theappropriate doses of chemotherapeutic agents and/or hormone therapeuticagent will be approximately those already employed in clinical therapieswherein the chemotherapeutic agent and/or hormone therapeutic agent areadministered alone or in combination with other chemotherapeutic agents.Variation in dosage will likely occur depending on the condition beingtreated. As described above, in some embodiments, the chemotherapeuticagents and/or hormone therapeutic agents may be administered at areduced level.

The dose of the rapamycin or its derivative therein in the nanoparticlecomposition will vary with the nature of the combination therapy and theparticular disease being treated. In some embodiments, the amount ofrapamycin or a derivative thereof in the nanoparticle composition in thecombination therapy is included in any of the following ranges: about0.5 to about 5 mg, about 5 to about 10 mg, about 10 to about 15 mg,about 15 to about 20 mg, about 20 to about 25 mg, about 20 to about 50mg, about 25 to about 50 mg, about 50 to about 75 mg, about 50 to about100 mg, about 75 to about 100 mg, about 100 to about 125 mg, about 125to about 150 mg, about 150 to about 175 mg, about 175 to about 200 mg,about 200 to about 225 mg, about 225 to about 250 mg, about 250 to about300 mg, about 300 to about 350 mg, about 350 to about 400 mg, about 400to about 450 mg, or about 450 to about 500 mg. In some embodiments, theamount of rapamycin or derivative thereof in the effective amount of thenanoparticle composition (e.g., a unit dosage form) for combinationtherapy is in the range of about 54 mg to about 540 mg, such as about180 mg to about 270 mg or about 216 mg. In some embodiments, theconcentration of the rapamycin in the nanoparticle composition for usein combination therapy is dilute (about 0.1 mg/ml) or concentrated(about 100 mg/ml), including for example any of about 0.1 to about 50mg/ml, about 0.1 to about 20 mg/ml, about 1 to about 10 mg/ml, about 2mg/ml to about 8 mg/ml, about 4 to about 6 mg/ml, about 5 mg/ml. In someembodiments, the concentration of the rapamycin or a derivative thereofin the nanoparticle composition in combination therapy is at least aboutany of 0.5 mg/ml, 1.3 mg/ml, 1.5 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml,25 mg/ml, 30 mg/ml, 40 mg/ml, or 50 mg/ml.

Exemplary effective amounts of rapamycin or a derivative thereof in thenanoparticle composition for use in combination therapy include, but arenot limited to, about any of 25 m g/m², 30 mg/m², 50 mg/m², 60 mg/m², 75mg/m², 80 mg/m², 90 mg/m², 100 mg/m², 120 mg/m², 160 mg/m², 175 mg/m²,180 mg/m², 200 mg/m², 210 mg/m², 220 mg/m², 250 mg/m², 260 mg/m², 300mg/m², 350 mg/m², 400 mg/m², 500 mg/m², 540 mg/m², 750 mg/m², 1000mg/m², or 1080 mg/m² rapamycin. In various embodiments, the rapamycin ora derivative thereof in the nanoparticle composition includes less thanabOut any of 350 mg/m², 300 mg/m², 250 mg/m², 200 mg/m², 150 mg/m², 120m g/m², 100 mg/m², 90 mg/m², 50 mg/m², or 30 mg/m² rapamycin or aderivative thereof. In some embodiments, the amount of the rapamycin ora derivative thereof per administration in combination therapy is lessthan about any of 25 mg/m², 22 mg/m², 20 mg/m², 18 mg/m², 15 mg/m², 14mg/m², 13 mg/m², 12 mg/m², 11 mg/m², 10 mg/m², 9 mg/m², 8 mg/m², 7mg/m², 6 mg/m², 5 mg/m², 4 mg/m², 3 mg/m², 2 mg/m², or 1 mg/m². In someembodiments, the effective amount of rapamycin or a derivative thereofin the composition for use in combination therapy is included in any ofthe following ranges: about 1 to about 5 mg/m², about 5 to about 10mg/m², about 10 to about 25 mg/m², about 25 to about 50 mg/m², about 50to about 75 mg/m², about 75 to about 100 mg/m², about 100 to about 125mg/m², about 125 to about 150 mg/m², about 150 to about 175 mg/m², about175 to about 200 mg/m², about 200 to about 225 mg/m², about 225 to about250 mg/m², about 250 to about 300 mg/m², about 300 to about 350 mg/m²,or about 350 to about 400 mg/m². Preferably, the effective amount ofrapamycin or a derivative thereof in the composition for use incombination therapy is about 30 to about 300 mg/m², such as about 100 toabout 150 mg/m², about 120 mg/m², about 130 mg/m², or about 140 mg/m².

In some embodiments of any of the above aspects, the effective amount ofrapamycin or a derivative thereof in the nanoparticle composition foruse in combination therapy includes at least about any of 1 mg/kg, 2.5mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg, or 20 mg/kg. In variousembodiments, the effective amount of rapamycin or a derivative thereofin the nanoparticle composition for use in combination therapy includesless than about any of 350 mg/kg, 300 mg/kg, 250 mg/kg, 200 mg/kg, 150mg/kg, 100 mg/kg, 50 mg/kg, 25 mg/kg, 20 mg/kg, 10 mg/kg, 5 mg/kg, or 1mg/kg rapamycin or a derivative thereof.

Exemplary dosing frequencies of rapamycin or a derivative thereof in thenanoparticle composition for use in combination therapy include, but arenot limited to, weekly without break; weekly, three out of four weeks;once every three weeks; once every two weeks; weekly, two out of threeweeks. In some embodiments, the rapamycin or a derivative thereof in thenanoparticle composition is administered in combination about once every2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, oronce every 8 weeks. In some embodiments, the rapamycin or a derivativethereof in the nanoparticle composition is administered in combinationtherapy at least about any of 1×, 2×, 3×, 4×, 5×, 6×, or 7× (i.e.,daily) a week. In some embodiments, the intervals between eachadministration in combination therapy are less than about any of 6months, 3 months, 1 month, 20 days, 15, days, 12 days, 10 days, 9 days,8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. Insome embodiments, the intervals between each administration incombination therapy are more than about any of 1 month, 2 months, 3months, 4 months, 5 months, 6 months, 8 months, or 12 months. In someembodiments, there is no break in the dosing schedule. In someembodiments, the interval between each administration is no more thanabout a week.

The administration of the rapamycin or a derivative thereof in thenanoparticle composition in combination therapy can be extended over anextended period of time, such as from about a month up to about sevenyears. In some embodiments, the rapamycin or a derivative thereof in thenanoparticle composition is administered over a period of at least aboutany of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 48, 60, 72,or 84 months. In some embodiments, the rapamycin or derivative thereofnanoparticle composition is administered over a period of at least onemonth, wherein the interval between each administration is no more thanabout a week, and wherein the dose of the rapamycin or a derivativethereof in the nanoparticle composition at each administration is about0.25 mg/m² to about 75 mg/m², such as about 0.25 mg/m² to about 25 mg/m²or about 25 mg/m² to about 50 mg/m².

In some embodiments, the dosage of rapamycin in the nanoparticlecomposition in the combination therapy can be in the range of 100-400mg/m² when given on a 3 week schedule, or 50-250 mg/m² when given on aweekly schedule. Preferably, the amount of rapamycin is about 80 toabout 180 mg/m² (e.g., about 100 mg/m² to about 150 mg/m², such as about120 mg/m²).

Other exemplary dosing schedules for the administration of thenanoparticle composition (e.g., rapamycin/albumin nanoparticlecomposition) in combination therapy include, but are not limited to, 100mg/m², weekly, without break; 75 mg/m² weekly, 3 out of four weeks; 100mg/m², weekly, 3 out of 4 weeks; 125 mg/m², weekly, 3 out of 4 weeks;125 mg/m², weekly, 2 out of 3 weeks; 130 mg/m², weekly, without break;175 mg/m², once every 2 weeks; 260 mg/m², once every 2 weeks; 260 mg/m²,once every 3 weeks; 180-300 mg/m², every three weeks; 60-175 mg/m²,weekly, without break; 20-150 mg/m² twice a week; and 150-250 mg/m²twice a week. The dosing frequency of the composition may be adjustedover the course of the treatment based on the judgment of theadministering physician.

The rapamycin or a derivative thereof nanoparticle compositionsdescribed herein can be administered to an individual (such as human)during combination therapy via various routes, such as parenterally,including intravenous, intra-arterial, intraperitoneal, intrapulmonary,oral, inhalation, intravesicular, intramuscular, intra-tracheal,subcutaneous, intraocular, intrathecal, or transdermal. For example, thenanoparticle composition can be administered by inhalation to treatconditions of the respiratory tract. The rapamycin or a derivativethereof nanoparticle compositions can be used to treat respiratoryconditions such as pulmonary fibrosis, broncheolitis obliterans, lungcancer, bronchoalveolar carcinoma, and the like. In some embodiments,the nanoparticle compositions is administrated intravenously. In someembodiments, the nanoparticle compositions is administered orally.

In some embodiments, the nanoparticle composition of the rapamycin or aderivative thereof and the chemotherapeutic agent is administeredaccording to any of the dosing regimes described in Table 1.

In some embodiments, there is provided a method of treating cancer in anindividual comprising administering to the individual: a) an effectiveamount of a composition comprising nanoparticles comprising a rapamycinor a derivative thereof and an albumin, and b) an effective amount of atleast one other chemotherapeutic agent as provided in Rows 1 to 53 inTable 1. In some embodiments, the administration of the nanoparticlecomposition and the chemotherapeutic agent may be any of the dosingregimes as indicated in Rows 1 to 53 in Table 1. In some embodiments,the cancer is early stage cancer, non-metastatic cancer, primary cancer,advanced cancer, locally advanced cancer, metastatic cancer, cancer inremission, recurrent cancer, cancer in an adjuvant setting, cancer in aneoadjuvant setting, or cancer substantially refractory to hormonetherapy. In some embodiments, the cancer is a solid tumor. In someembodiments, the cancer is not a solid tumor (i.e., other than a solidtumor). In some embodiments, the cancer is a plasmacytoma. In someembodiments, the cancer is multiple myeloma, renal cell carcinoma,prostate cancer, lung cancer, melanoma, brain cancer (e.g.,glioblastoma), ovarian cancer, or breast cancer. In some embodiments,the cancer is a carcinoma (i.e., other than a carcinoma). In someembodiments, the cancer is not colon cancer (i.e., other than coloncancer). In some embodiments, the cancer is not breast cancer (i.e.,other than breast cancer). In some embodiments, the cancer is notovarian cancer, prostate cancer, or brain cancer. In some embodiments,one or more symptoms of the cancer are ameliorated. In some embodiments,the cancer is delayed or prevented.

TABLE 1 Row No. Combination Regime/Dosage 1. RAPA + Carboplatin + RAPA:Rapamycin or a derivative thereof: any doses or Herceptin ® regimesdescribed above for combination therapy Carbo: AUC = 2 D 1, 8, 15 q4 wk× 6 Herceptin ®: 4 mg/kg on wk 1, 2 mg/kg all subsequent weeks 2. RAPAalone RAPA: Rapamycin or a derivative thereof: any doses or(+Herceptin ®) regimes described above for combination therapy 3. RAPA +Navelbine ® L1: Rapamycin or a derivative thereof: any doses or (±G-CSF)regimes described above for combination therapy Nav: 15 mg/m² L2:Rapamycin or a derivative thereof: any doses or regimes described abovefor combination therapy Nav: 20 mg/m² L3: Rapamycin or a derivativethereof: any doses or regimes described above for combination therapyNav: 22.5 mg/m² L4: Rapamycin or a derivative thereof: any doses orregimes described above for combination therapy Nav: 25 mg/m² L5:Rapamycin or a derivative thereof: any doses or regimes described abovefor combination therapy Nav: 25 mg/m² qwk all levels 4. RAPA + Xeloda ®RAPA: Rapamycin or a derivative thereof: any doses or regimes describedabove for combination therapy Xeloda ®: 825 mg/m² D 1-14 q3 wk 5. RAPA +Anthracycline 6. RAPA + RAPA: Rapamycin or a derivative thereof: anydoses or Gemcitabine regimes described above for combination therapyGem: 1000 mg/m2 qwk × ⅔ 7. RAPA + Lapatinib Rapamycin or a derivativethereof: any doses or regimes described above for combination therapyLapatinib: starting at 1000 mg/d × 2 days 8. RAPA + FEC RAPA: Rapamycinor a derivative thereof: any doses or (+Herceptin ®) regimes describedabove for combination therapy FEC: 4 cycles (+Herceptin ® for HER2+ pts)9. RAPA + Carboplatin + RAPA: Rapamycin or a derivative thereof: anydoses or Avastin ® regimes described above for combination therapyCarbo: AUC = 2 qwk D 1, 8, 15 Avastin ®: 10 mg/m² q2 wk 10. RAPA +Avastin ® RAPA: Rapamycin or a derivative thereof: any doses or regimesdescribed above for combination therapy + Avastin ® 11. RAPA +Xeloda ® + RAPA: Rapamycin or a derivative thereof: any doses orLapatinib regimes described above for combination therapy 12. RAPA +Gemcitabine RAPA: Rapamycin or a derivative thereof: any doses orregimes described above for combination therapy Gem: 1250 mg/m² D 1, 8q3 wk 13. RAPA + Sutent ® 14. RAPA + AC + G- AC + G-CSF q2 wk × 4followed by RAPA: CSF (+Herceptin ®) Rapamycin or a derivative thereof:any doses or regimes described above for combination therapy(+Herceptin ® for HER2+ pts) 15. RAPA + AC + G- Dose dense AC + G-CSFfollowed by RAPA: CSF (+Herceptin ®) Rapamycin or a derivative thereof:any doses or regimes described above for combination therapy(+Herceptin ® for HER2+ pts) 16. RAPA + AC AC followed by RAPA:Rapamycin or a derivative thereof: any doses or regimes described abovefor combination therapy 17. RAPA + AC AC q2 wk followed by RAPA:(+G-CSF) Rapamycin or a derivative thereof: any doses or regimesdescribed above for combination therapy Rx length 16 wks 18. RAPA + ACDose dense AC followed by RAPA: (+Avastin ®) Rapamycin or a derivativethereof: any doses or regimes described above for combination therapy(+Avastin ® in HER2+ pts) 19. RAPA + AC AC (such as about 60 mg/m²adriamycin and 600 mg/m² cyclophosphamide, once every two weeks)followed by RAPA: Rapamycin or a derivative thereof: any doses orregimes described above for combination therapy 20. RAPA + AC + ACfollowed by RAPA: Rapamycin or a derivative thereof: Neulasta ® anydoses or regimes described above for combination therapy 21. RAPA + FECRAPA: Rapamycin or a derivative thereof: any doses or (+Herceptin ®)regimes described above for combination therapy followed by 5-FU: 500mg/m² q3 wk Epirubicin: 100 mg/m² (without Herceptin ®) or Epirubicin:75 mg/m² (with Herceptin ® for HER2+ pts) Cyclophosphamide: 500 mg/m² q3wk 22. RAPA + Arm 1: Neoadjuvant: Gem: 2000 mg/m², RAPA: Rapamycin orGemcitabine + a derivative thereof: any doses or regimes described abovefor Epirubicin combination therapy, Epi 50 mg/m² q2 wk × 6 Arm 2:Adjuvant: Gem: 2000 mg/m², RAPA: Rapamycin or a derivative thereof: anydoses or regimes described above for combination therapy 23. RAPA +Herceptin ® + RAPA: Rapamycin or a derivative thereof: any doses orNavelbine regimes described above for combination therapy + Herceptin ®followed by Navelbine ® + Herceptin ® 24. RAPA + Carboplatin TAC vs ACfollowed by RAPA: (+Herceptin ®) + AC Rapamycin or a derivative thereof:any doses or regimes described above for combination therapy + carbo vsAC followed by RAPA: Rapamycin or a derivative thereof: any doses orregimes described above for combination therapy + carbo + Herceptin ®25. RAPA + RAPA: Rapamycin or a derivative thereof: any doses orCapecitabine regimes described above for combination therapy Xeloda ®850 mg/m² D 1-14 q3 wk × 4 26. RAPA + Carboplatin RAPA: Rapamycin or aderivative thereof: any doses or (+Avastin ®) regimes described abovefor combination therapy Carbo qwk + Avastin ® in HER2+ pts 27. RAPA +Carboplatin + RAPA: Rapamycin or a derivative thereof: any doses orHerceptin ® + Avastin ® regimes described above for combination therapyCarbo: AUC = 5 + Herceptin ® + Avastin ® 4 week cycle × 6 28. RAPA +Lapatinib RAPA: Rapamycin or a derivative thereof: any doses or regimesdescribed above for combination therapy Lapatinib: 1000 mg/day 29.RAPA + RAPA: Rapamycin or a derivative thereof: any doses orCapecitabine regimes described above for combination therapy Xeloda ®:1000 mg/m² D 1-14 q3 wk × 4 30. RAPA ± Avastin ® + RAPA: Rapamycin or aderivative thereof: any doses or AC (+G-CSF) regimes described above forcombination therapy ± Avastin ® followed by A qwk + C daily 31. RAPA +AC RAPA: Rapamycin or a derivative thereof: any doses or regimesdescribed above for combination therapy followed by AC 32. RAPA +Carboplatin + RAPA: Rapamycin or a derivative thereof: any doses orAvastin ® regimes described above for combination therapy Carbo: AUC = 6q3 wk Avastin ®: 15 mg/kg 4 cycles 33. RAPA + Carboplatin RAPA:Rapamycin or a derivative thereof: any doses or regimes described abovefor combination therapy Carbo fixed at AUC = 6 q3 wk 34. RAPA +Carboplatin + Avastin ® 35. RAPA + Gemcitabine or RAPA + Avastin ® 36.RAPA + Carboplatin + RAPA: Rapamycin or a derivative thereof: any dosesor Avastin ® regimes described above for combination therapy Carbo: AUC= 6 q3 wk + Avastin ® 37. RAPA + Alimta ® RAPA: Rapamycin or aderivative thereof: any doses or regimes described above for combinationtherapy Pemetrexed: 500 mg q3 wk 38. RAPA + Cisplatin 39. RAPA +Navelbine ® + Cisplatin 40. RAPA + Carboplatin RAPA: Rapamycin or aderivative thereof: any doses or regimes described above for combinationtherapy Carbo: AUC = 6 q3 wk 41. RAPA + Carboplatin RAPA: Rapamycin or aderivative thereof: any doses or regimes described above for combinationtherapy Carbo: AUC = 6 42. RAPA + Avastin ® RAPA: Rapamycin or aderivative thereof: any doses or regimes described above for combinationtherapy Avastin ®: 10 mg/m² q2 wk 43. RAPA + 5-FU + RAPA: Rapamycin or aderivative thereof: any doses or Cisplatin regimes described above forcombination therapy 5-FU: 750 mg/m² CIV × 5 cisplatin: 75 mg/m² D 1followed by XRT/surgery 44. RAPA + Cetuximab 45. RAPA + Satraplatin 46.RAPA + RAPA: Rapamycin or a derivative thereof: any doses or Gemcitabineregimes described above for combination therapy Gemcitabine: 1000 mg/m²D 1 and D 8 47. RAPA + Gefitinib RAPA: Rapamycin or a derivativethereof: any doses or regimes described above for combination therapyGefitinib starting at 1000 mg/d × 2 48. RAPA + Sorafenib + RAPA:Rapamycin or a derivative thereof: any doses or Carboplatin regimesdescribed above for combination therapy Sorafenib: D 2-19 Carbo: AUC = 6D 1 49. RAPA + RAPA: Rapamycin or a derivative thereof: any doses orCapecitabine regimes described above for combination therapy + Xeloda ®at a range of about 500-2500 mg/m² (such as any of about 550 mg/m², 650mg/m², 85 mg/m², 850 mg/m², 100 mg/m², 1250 mg/m²) 50. RAPA + WeeklyGemcitabine 51. RAPA + anti- angiogenic agent(s) 52. RAPA + proteasomeinhibitor(s) 53. RAPA + tyrosine kinase inhibitor(s) 54. RAPA + EGFRinhibitor(s)

As used in herein (for example in Table 1), RAPA refers to a compositioncomprising nanoparticles comprising rapamycin or a derivative thereofand a carrier protein (e.g., albumin); GW572016 refers to lapatinib; Xelrefers to capecitabine or Xeloda®; bevacizumab is also known asAvastin®; trastuzumab is also known as Herceptin®; pemetrexed is alsoknown as Alimta®; cetuximab is also known as Erbitux®; gefitinib is alsoknown as Iressa®; FEC refers to a combination of 5-fluorouracil,Epirubicin and Cyclophosphamide; AC refers to a combination ofAdriamycin plus Cyclophosphamide.

As used herein (for example in Table 1), AUC refers to area under curve;q4wk refers to a dose every 4 weeks; q3wk refers to a dose every 3weeks; q2wk refers to a dose every 2 weeks; qwk refers to a weekly dose;qwk×3/4 refers to a weekly dose for 3 weeks with the 4^(th) week off;qwk×2/3 refers to a weekly dose for 2 weeks with the 3^(rd) week off.

In some embodiments, the present invention provides a method of treatingcancer comprising a first therapy comprising administering nanoparticlescomprising rapamycin or a derivative thereof and a carrier protein(e.g., albumin) and a second therapy comprising surgery, radiation, genetherapy, immunotherapy, bone marrow transplantation, stem celltransplantation, targeted therapy, cryotherapy, ultrasound therapy,and/or photodynamic therapy. In some embodiments, the method comprisesa) a first therapy comprising administering to the individual acomposition comprising nanoparticles of rapamycin and an albumin; and b)a second therapy comprising surgery, radiation, gene therapy,immunotherapy, bone marrow transplantation, stem cell transplantation,targeted therapy, cryotherapy, ultrasound therapy, and/or photodynamictherapy. In some embodiments, the cancer may be prostate cancer. In someembodiments, the second therapy is radiation therapy. In someembodiments, the second therapy is surgery.

The administration of the rapamycin or a derivative thereof nanoparticlecomposition may be prior to the radiation and/or surgery, after theradiation and/or surgery, or concurrent with the radiation and/orsurgery. For example, the administration of the nanoparticle compositionmay precede or follow the radiation and/or surgery therapy by intervalsranging from minutes to weeks. In some embodiments, the time periodbetween the first and the second therapy is such that the rapamycin or aderivative thereof nanoparticles and the radiation/surgery would stillbe able to exert an advantageously combined effect on the cell. Forexample, the rapamycin or derivative thereof in the nanoparticlecomposition may be administered less than about any of 1, 3, 6, 9, 12,18, 24, 48, 60, 72, 84, 96, 108, 120 hours prior to the radiation and/orsurgery. In some embodiments, the nanoparticle composition isadministered less than about 9 hours prior to the radiation and/surgery.In some embodiments, the nanoparticle composition is administered lessthan about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days prior to theradiation/surgery. In some embodiments, the rapamycin or a derivativethereof in the nanoparticle composition is administered less than aboutany of 1, 3, 6, 9, 12, 18, 24, 48, 60, 72, 84, 96, 108, or 120 hoursafter the radiation and/or surgery. In some embodiments, it may bedesirable to extend the time period for treatment significantly, whereseveral days to several weeks lapse between the two therapies.

Radiation contemplated herein includes, for example, γ-rays, X-rays(external beam), and the directed delivery of radioisotopes to tumorcells. Other forms of DNA damaging factors are also contemplated such asmicrowaves and UV irradiation are also contemplated. Radiation may begiven in a single dose or in a series of small doses in adose-fractionated schedule. The amount of radiation contemplated hereinranges from about 1 to about 100 Gy, including, for example, about 5 toabout 80, about 10 to about 50 Gy, or about 10 Gy. The total dose may beapplied in a fractioned regime. For example, the regime may comprisefractionated individual doses of 2 Gy. Dosage ranges for radioisotopesvary widely, and depends on the half-life of the isotope and thestrength and type of radiation emitted.

When the radiation comprises use of radioactive isotopes, the isotopemay be conjugated to a targeting agent, such as a therapeutic antibody,which carries the radionucleotide to the target tissue. Suitableradioactive isotopes include, but are not limited to, astatine²¹¹,carbon¹⁴, chromium⁵¹, chlorine³⁶, iron⁵⁷, cobalt⁵⁸, copper⁶⁷, Eu¹⁵²,gallium⁶⁷, hydrogen³, iodine¹²³, iodine¹³¹, indium¹¹¹, iron⁵⁹,phosphorus³², rhenium¹⁸⁶, selenium⁷⁵, sulphur³⁵, technicium^(99m),and/or yttrium⁹⁰.

In some embodiments, enough radiation is applied to the individual so asto allow reduction of the normal dose of the rapamycin or a derivativethereof in the nanoparticle composition required to affect the samedegree of treatment by at least about any of 5%, 10%, 20%, 30%, 50%,60%, 70%, 80%, 90%, or more. In some embodiments, enough rapamycin orderivative thereof in the nanoparticle composition is administered so asto allow reduction of the normal dose of the radiation required toaffect the same degree of treatment by at least about any of 5%, 10%,20%, 30%, 50%, 60%, 70%, 80%, 90%, or more. In some embodiments, thedose of both the rapamycin or a derivative thereof in the nanoparticlecomposition and the radiation are reduced as compared to thecorresponding normal dose of each when used alone.

In some embodiments, the combination of administration of the rapamycinor a derivative thereof nanoparticle composition and the radiationtherapy produce supra-additive effect. In some embodiments, therapamycin or a derivative thereof in the nanoparticle composition isadministered once at the dose of about 50 mg to 540 mg or about 30 mg/m²to 300 mg/m², and the radiation is applied five times at 80 Gy daily.

Administration of rapamycin or a derivative thereof nanoparticlecompositions disclosed above in conjunction with administration ofchemotherapeutic agent and/or hormone therapeutic agent is equallyapplicable to those in conjunction with radiation therapy and/orsurgery.

In some embodiments, the nanoparticle composition of the rapamycin or aderivative thereof nanoparticles and/or the chemotherapeutic agent isadministered in conjunction with radiation according to any of thedosing regimes described in Table 2.

In some embodiments, there is provided a method of treating cancer in anindividual comprises a) a first therapy comprising administering to theindividual a composition comprising nanoparticles comprising rapamycinor a derivative thereof and an albumin; and b) a second therapycomprising radiation as provided in Rows 1 to 11 in Table 2. In someembodiments, the administration of the nanoparticle composition and thechemotherapeutic agent may be any of the dosing regimes as indicated inRows 1 to 11 in Table 2. In some embodiments, the cancer is early stagecancer, non-metastatic cancer, primary cancer, advanced cancer, locallyadvanced cancer, metastatic cancer, cancer in remission, recurrentcancer, cancer in an adjuvant setting, cancer in a neoadjuvant setting,or cancer substantially refractory to hormone therapy. In someembodiments, the cancer is a solid tumor. In some embodiments, thecancer is not a solid tumor (i.e., other than a solid tumor). In someembodiments, the cancer is a plasmacytoma. In some embodiments, thecancer is multiple myeloma, renal cell carcinoma, prostate cancer, lungcancer, melanoma, brain cancer (e.g., glioblastoma), ovarian cancer, orbreast cancer. In some embodiments, the cancer is a carcinoma (i.e.,other than a carcinoma). In some embodiments, the cancer is not coloncancer (i.e., other than colon cancer). In some embodiments, the canceris not breast cancer (i.e., other than breast cancer). In someembodiments, the cancer is not ovarian cancer, prostate cancer, or braincancer.

TABLE 2 Row No. Combination Regime/Dosage 1 RAPA + Radiation 2 RAPA +Carboplatin + Radiation 3 RAPA + Carboplatin + 1 cycle RAPA/Carboinduction followed by Radiation 2 or 3 times weekly pulse RAPA +radiation 4 RAPA + Carboplatin + Radiation 5 RAPA + Carboplatin + RAPA:Rapamycin or a derivative thereof: Radiation any doses or regimesdescribed above for combination therapy + carbo + radiation followed byRAPA: Rapamycin or a derivative thereof: any doses or regimes describedabove for combination therapy + carbo 6 RAPA + Radiation 7 RAPA +Cetuximab + Radiation 8 RAPA + Carboplatin + Induction: RAPA: Rapamycinor a derivative 5-FU + Hydroxyurea + thereof: any doses or regimesdescribed above Radiation for combination therapy + carbo: AUC = 2followed by Concurrent chemoradiation: RAPA: Rapamycin or a derivativethereof: any doses or regimes described above for combination therapy;5-FU: 600 mg/m²; hydroxyurea: 5000 mg BID 9 RAPA + Carboplatin + RAPA:Rapamycin or a derivative thereof: Erbitux ® + Radiation any doses orregimes described above for combination therapy Eribitux ®: 400 mg/m²day 7, 250 mg/m² qwk × 7 Carbo: AUC = 1.5 qwk × 7 IMRT 10 RAPA +Gemcitabine + Qwk Radiation 11 RAPA + Cisplatin + Radiation

Metronomic Therapy Regimes

The present invention also provides metronomic therapy regimes for anyof the methods of treatment and methods of administration describedherein. Exemplary metronomic therapy regimes and embodiments for the useof metronomic therapy regimes are discussed below and disclosed in U.S.Ser. No. 11/359,286, filed Feb. 21, 2006, published as U.S. Pub. No.2006/0263434 (such as those described in paragraphs [0138] to [0157]),which is hereby incorporated by reference in its entirety. In someembodiments, the nanoparticle composition is administered over a periodof at least one month, wherein the interval between each administrationis no more than about a week, and wherein the dose of rapamycin or aderivative thereof at each administration is about 0.25% to about 25% ofits maximum tolerated dose following a traditional dosing regime. Insome embodiments, the nanoparticle composition is administered over aperiod of at least two months, wherein the interval between eachadministration is no more than about a week, and wherein the dose ofrapamycin or a derivative thereof at each administration is about 1% toabout 20% of its maximum tolerated dose following a traditional dosingregime. In some embodiments, the dose of rapamycin or a derivativethereof per administration is less than about any of 25%, 24%, 23%, 22%,20%, 18%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,or 1% of the maximum tolerated dose. In some embodiments, thenanoparticle composition is administered at least about any of 1×, 2×,3×, 4×, 5×, 6×, or 7× (i.e., daily) a week. In some embodiments, theintervals between each administration are less than about any of 6months, 3 months, 1 month, 20 days, 15, days, 12 days, 10 days, 9 days,8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. Insome embodiments, the intervals between each administration are morethan about any of 1 month, 2 months, 3 months, 4 months, 5 months, 6months, 8 months, or 12 months. In some embodiments, the composition isadministered over a period of at least about any of 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 18, 24, 30, 36, 48, 60, 72, or 84 months.

Pharmaceutical Agents

Provided herein are compositions comprising nanoparticles that compriserapamycin for use in the methods of treatment of cancer, methods ofadministration, and dosing regimes described herein. In someembodiments, rapamycin may be rapamycin or its derivatives orpharmaceutically acceptable salts and accordingly the inventioncontemplates and includes all these embodiments. Rapamycin is sometimesreferred to elsewhere as sirolimus, rapammune, or rapamune. Derivativesof rapamycin include, but are not limited to, compounds that arestructurally similar to rapamycin or are in the same general chemicalclass as rapamycin.

In some embodiments, the derivative of rapamycin retains one or moresimilar biological, pharmacological, chemical and/or physical properties(including, for example, functionality) as rapamycin. In someembodiments, the rapamycin derivative has at least about any of 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of an activity ofrapamycin. For example, the decrease in the size of a tumor, the numberof cancer cells, or the growth rate of a tumor caused by a rapamycinderivative is preferably at least about any of 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% or 100% of the corresponding decrease caused bythe same amount of rapamycin. An exemplary rapamycin derivative includesbenzoyl rapamycin, such as that disclosed in paragraph [0022] of WO2006/089207, which is hereby incorporated by reference in its entirety.Other exemplary rapamycin derivatives include WY-090217, AY-22989,NSC-226080, SiiA-9268A, oxaazacyclohentriacontine, temsirolimus (CCl-779(Wyeth)), everolimus (RAD001 (Novartis)), pimecrolimus (ASM981),SDZ-RAD, SAR943, ABT-578, AP23573, and Biolimus A9.

Carrier Proteins

Provide herein are compositions comprising nanoparticles that compriserapamycin and a carrier protein for use methods of treatment of cancer,methods of administration, and dosage regimes described herein. In someembodiments, rapamycin may be rapamycin or its derivatives orpharmaceutically acceptable salts and accordingly the inventioncontemplates and includes all these embodiments. In some embodiments,the carrier protein is albumin. In some embodiments, the carrier proteinis human serum albumin.

Examples of suitable carrier proteins include proteins normally found inblood or plasma, which include, but are not limited to, albumin,immunoglobulin including IgA, lipoproteins, apolipoprotein B, α-acidglycoprotein, β-2-macroglobulin, thyroglobulin, transferin, fibronectin,factor VII, factor VIII, factor IX, factor X, and the like. In someembodiments, the carrier protein is a non-blood protein, such as casein,α-lactalbumin, or β-lactoglobulin. The carrier proteins may either benatural in origin or synthetically prepared. In some embodiments, thepharmaceutical acceptable carrier comprises albumin, such as human serumalbumin (HSA). HSA is a highly soluble globular protein of M_(r) 65K andconsists of 585 amino acids. HSA is the most abundant protein in theplasma and accounts for 70-80% of the colloid osmotic pressure of humanplasma. The amino acid sequence of HSA contains a total of 17 disulphidebridges, one free thiol (Cys 34), and a single tryptophan (Trp 214).Other albumins are contemplated, such as bovine serum albumin. Use ofsuch non-human albumins could be appropriate, for example, in thecontext of use of these compositions in non-human mammals, such as theveterinary animals (including domestic pets and agricultural animals).

Human serum albumin (HSA) has multiple hydrophobic binding sites (atotal of eight for fatty acids, an endogenous ligand of HSA) and binds adiverse set of drugs, especially neutral and negatively chargedhydrophobic compounds (Goodman et al., The Pharmacological Basis ofTherapeutics, 9^(th) ed, McGraw-Hill New York (1996)). Two high affinitybinding sites have been proposed in subdomains IIA and IIIA of HSA,which are highly elongated hydrophobic pockets with charged lysine andarginine residues near the surface which function as attachment pointsfor polar ligand features (see, e.g., Fehske et al., Biochem. Pharmcol.,30, 687-92 (1981), Vorum, Dan. Med. Bull., 46, 379-99 (1999),Kragh-Hansen, Dan. Med. Bull., 1441, 131-40 (1990), Curry et al., Nat.Struct. Biol., 5, 827-35 (1998), Sugio et al., Protein. Eng., 12, 439-46(1999), He et al., Nature, 358, 209-15 (1992), and Carter et al., Adv.Protein. Chem., 45, 153-203 (1994)).

The carrier protein (e.g., albumin) in the composition generally servesas a carrier for rapamycin or derivative thereof, i.e., the carrierprotein in the composition makes the rapamycin or derivative thereofmore readily suspendable in an aqueous medium or helps maintain thesuspension as compared to compositions not comprising a carrier protein.This can avoid the use of toxic solvents for solubilizing rapamycin or aderivative thereof, and thereby can reduce one or more side effects ofadministration of rapamycin or a derivative thereof into an individual(e.g., human). In some embodiments, the composition is substantiallyfree (e.g. free) of organic solvents or surfactants. A composition is“substantially free of organic solvent” or “substantially free ofsurfactant” if the amount of organic solvent or surfactant in thecomposition is not sufficient to cause one or more side effect(s) in anindividual when the composition is administered to the individual.

Rapamycin is “stabilized” in an aqueous suspension if it remainssuspended in an aqueous medium (e.g., without visible precipitation orsedimentation) for an extended period of time, such as for at leastabout any of 0.1, 0.2, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,24, 36, 48, 60, or 72 hours. The suspension is generally, but notnecessarily, suitable for administration to an individual (e.g., human).Stability of the suspension is generally (but not necessarily) evaluatedat storage temperature, such as room temperature (e.g., 20-25° C.) orrefrigerated conditions (e.g., 4° C.). For example, a suspension isstable at a storage temperature if it exhibits no flocculation orparticle agglomeration visible to the naked eye or when viewed under theoptical microscope at 1000 times, at about fifteen minutes afterpreparation of the suspension. Stability can also be evaluated underaccelerated testing conditions, such as at a temperature that is higherthan about 40° C.

In some embodiments, the composition comprises nanoparticles comprising(in various embodiments consisting essentially of) rapamycin and acarrier protein. When rapamycin is in a liquid form, the particles ornanoparticles are also referred to as droplets or nanodroplets. In someembodiments, rapamycin is coated with the carrier protein. Particles(such as nanoparticles) of poorly water soluble pharmaceutical agentshave been disclosed in, for example, U.S. Pat. Nos. 5,916,596;6,506,405; and 6,537,579 and also in U.S. Pat. App. Pub. No.2005/0004002A1.

The amount of carrier protein in the composition described herein willvary depending on the rapamycin or derivative thereof and othercomponents in the composition. In some embodiments, the compositioncomprises a carrier protein in an amount that is sufficient to stabilizethe rapamycin in an aqueous suspension, for example, in the form of astable colloidal suspension (e.g., a stable suspension ofnanoparticles). In some embodiments, the carrier protein is in an amountthat reduces the sedimentation rate of rapamycin in an aqueous medium.For particle-containing compositions, the amount of the carrier proteinalso depends on the size and density of particles of rapamycin.

In some embodiments of any of the aspects of the invention, therapamycin or a derivative thereof is coated with a carrier protein, suchas albumin (e.g., human serum albumin). In various embodiments, thecomposition comprises more than about any of 50%, 60%, 70%, 80%, 90%,95%, or 99% of the rapamycin or derivative thereof in nanoparticle form.In some embodiments, the rapamycin or derivative thereof constitutesmore than about any of 50%, 60%, 70%, 80%, 90%, 95%, or 99% of thenanoparticles by weight. In some embodiments, the nanoparticle has anon-polymeric matrix. In some embodiments, the rapamycin or derivativethereof is in an anhydrous, amorphous, and/or non-crystalline form. Insome embodiments, the rapamycin or derivative thereof is amorphous. Insome embodiments, the nanoparticles comprise a core of rapamucin orderivative thereof that is substantially free of polymeric materials(such as polymeric matrix).

In some embodiments, the albumin to rapamycin weight ratio in thenanoparticles or in the nanoparticle composition is about any of 18:1 orless, 15:1 or less, 14:1 or less, 13:1 or less, 12:1 or less, 11:1 orless, 10:1 or less, 9:1 or less, 8:1 or less, 7.5:1 or less, 7:1 orless, 6:1 or less, 5:1 or less, 4:1 or less, or 3:1 or less. In someembodiments, the composition comprises a stable aqueous suspension ofparticles (e.g., nanoparticles) comprising rapamycin or a derivativethereof and albumin (e.g., particles of rapamycin or a derivativethereof coated with albumin).

In some embodiments, the composition comprises nanoparticles of anyshape (e.g., a spherical or non-spherical shape) with an average or meandiameter of no greater than about 1000 nanometers (nm), such as nogreater than about any of 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400nm, 300 nm, 200 nm, or 100 nm. In some embodiments, the average or meandiameter of the particles is no greater than about 200 nm. In someembodiments, the average or mean diameter of the particles is betweenabout 20 to about 400 nm. In some embodiments, the average or meandiameter of the particles is between about 40 to about 200 nm. In someembodiments, the particles are sterile-filterable.

In some embodiments, the nanoparticles comprise the rapamycin or aderivative thereof coated with a coating comprising the carrier protein(such as albumin). In some embodiments, the coating consists essentiallyof or consists of the carrier protein. In some embodiments, at least aportion of the carrier protein in the nanoparticle portion of therapamycin (or rapamycin derivative) nanoparticle composition iscrosslinked (for example crosslinked by disulfide bonds).

The nanoparticles described herein may be present in a dry formulation(e.g., lyophilized composition) or suspended in a biocompatible medium.Suitable biocompatible media include, but are not limited to, water,buffered aqueous media, saline, buffered saline, optionally bufferedsolutions of amino acids, optionally buffered solutions of proteins,optionally buffered solutions of sugars, optionally buffered solutionsof vitamins, optionally buffered solutions of synthetic polymers,lipid-containing emulsions, and the like.

In some embodiments, the nanoparticles do not comprise a blood-insolublegas or do not comprise gas-filled microbubbles.

The amount of carrier protein in the composition described herein willvary depending on the rapamycin or derivative thereof and othercomponents in the composition. In some embodiments, the compositioncomprises a carrier protein in an amount that is sufficient to stabilizethe rapamycin in an aqueous suspension, for example, in the form of astable colloidal suspension (e.g., a stable suspension ofnanoparticles). In some embodiments, the carrier protein is in an amountthat reduces the sedimentation rate of rapamycin in an aqueous medium.The amount of the carrier protein also depends on the size and densityof particles of rapamycin.

Also provided herein are methods of reducing side effects associatedwith administration of a poorly water soluble pharmaceutical agent to ahuman, comprising administering to a human a pharmaceutical compositioncomprising the poorly water soluble pharmaceutical agent, and abiocompatible polymer (such as a carrier protein). For example, theinvention provides methods of reducing various side effects associatedwith administration of the poorly water soluble pharmaceutical agent,including, but not limited to, myelosuppression, neurotoxicity,hypersensitivity, inflammation, venous irritation, phlebitis, pain, skinirritation, neutropenic fever, anaphylactic reaction, hematologictoxicity, and cerebral or neurologic toxicity, and combinations thereof.In some embodiments, there is provided a method of reducinghypersensitivity reactions associated with administration of rapamycinor a derivative thereof, including, for example, severe skin rashes,hives, flushing, dyspnea, tachycardia, cancer (e.g., lymphoma); chestpain; black, tarry stools; general feeling of illness, shortness ofbreath; swollen glands; weight loss; yellow skin and eyes, abdominalpain; unexplained anxiousness; bloody or cloudy urine; bone pain;chills; confusion; convulsions (seizures); cough; decreased urge tourinate; fast, slow, or irregular heartbeat; fever; frequent urge tourinate; increased thirst; loss of appetite; lower back or side pain;mood changes; muscle pain or cramps; nausea or vomiting; numbness ortingling around lips, hands, or feet; painful or difficult urination;rash; sore throat; sores or white spots on lips or in mouth; swelling ofhands, ankles, feet, or lower legs; swollen glands; trouble breathing;unusual bleeding or bruising; unusual tiredness or weakness; weakness orheaviness of legs, skin ulcer or sores, weight gain, acne; constipation;diarrhea; difficulty in moving; headache; loss of energy or weakness;muscle pain or stiffness; pain; shaking or trembling; trouble sleeping;nosebleed; and/or swelling of the face. These side effects, however, aremerely exemplary and other side effects, or combination of side effects,associated with rapamycin can be reduced. The side effects may beimmediate or delayed (such as not occurring for a few days, weeks,months, or years after treatment begins).

Antimicrobial Agents in Compositions

In some embodiments, the compositions of the invention also includes anantimicrobial agent (e.g., an agent in addition to the rapamycin orderivative thereof in an amount sufficient to significantly inhibit(e.g., delay, reduce, slow, and/or prevent) microbial growth in thecomposition for use in the methods of treatment, methods ofadministration, and dosage regimes described herein. Exemplary microbialagents and embodiments for the use of microbial agents are disclosed inU.S. Ser. No. 11/514,030, filed Aug. 30, 2006 (such as those describedin paragraphs [0036] to [0058]). In some embodiments, the antimicrobialagent is a chelating agent, such as EDTA, edetate, citrate, pentetate,tromethamine, sorbate, ascorbate, derivatives thereof, or mixturesthereof. In some embodiments, the antimicrobial agent is a polydentatechelating agent. In some embodiments, the antimicrobial agent is anon-chelating agent, such as any of sulfites, benzoic acid, benzylalcohol, chlorobutanol, paraben, or derivatives thereof. In someembodiments, an antimicrobial other than rapamycin or derivativesthereof discussed above is not contained or used in the methods oftreatment, methods of administration, and dosage regimes describedherein

Sugar Containing Composition

In some embodiments, the compositions of the invention include a sugarfor use in the methods of treatment described herein. In someembodiments, the compositions of the invention include both a sugar andan antimicrobial agent for use in the methods of treatment describedherein. Exemplary sugars and embodiments for the use of sugars aredisclosed in U.S. Ser. No. 11/514,030, filed Aug. 30, 2006 (such asthose described in paragraphs [0084] to [0090]). In some embodiments,the sugar serves as a reconstitution enhancer which causes a lyophilizedcomposition to dissolve or suspend in water and/or aqueous solution morequickly than the lyophilized composition would dissolve without thesugar. In some embodiments, the composition is a liquid (e.g., aqueous)composition obtained by reconstituting or resuspending a drycomposition. In some embodiments, the concentration of sugar in thecomposition is greater than about 50 mg/ml. In some embodiments, thesugar is in an amount that is effective to increase the stability of therapamycin or derivative thereof in the composition as compared to acomposition without the sugar. In some embodiments, the sugar is in anamount that is effective to improve filterability of the composition ascompared to a composition without the sugar.

The sugar-containing compositions described herein may further compriseone or more antimicrobial agents, such as the antimicrobial agentsdescribed herein or in U.S. Ser. No. 11/514,030, filed Aug. 30, 2006. Inaddition to one or more sugars, other reconstitution enhancers (such asthose described in U.S. Pat. App. Publication No. 2005/0152979, which ishereby incorporated by reference in its entirety) can also be added tothe compositions. In some embodiments, a sugar is not contained or usedin the methods of treatment, methods of administration, and dosageregimes described herein

Stabilizing Agents in Composition

In some embodiments, the compositions of the invention also include astabilizing agent for use in the methods of treatment, methods ofadministration, and dosage regimes described herein. In someembodiments, the compositions of the invention include an antimicrobialagent and/or a sugar and/or a stabilizing agent for use in the methodsof treatment, methods of administration, and dosage regimes describedherein. Exemplary stabilizing agents and embodiments for the use ofstabilizing agents are disclosed in U.S. Ser. No. 11/513,756, filed Aug.30, 2006 (such as those described in paragraphs [0038] to [0083] and[0107] to [0114]). The present invention in one of its embodimentsprovides for compositions and methods of preparation of rapamycin whichretain the desirable therapeutic effects and remain physically and/orchemically stable upon exposure to certain conditions such as prolongedstorage, elevated temperature, or dilution for parenteraladministration. The stabilizing agent includes, for example, chelatingagents (e.g., citrate, malic acid, edetate, or pentetate), sodiumpyrophosphate, and sodium gluconate. In one embodiment, the inventionprovides pharmaceutical formulations of rapamycin or a derivativethereof comprising citrate, sodium pyrophosphate, EDTA, sodiumgluconate, citrate and sodium chloride, and/or a derivative thereof. Inanother embodiment, the invention provides a composition of rapamycincomprising a surfactant, wherein the rapamycin used for preparing theformulation is in an anhydrous form prior to being incorporated into thecomposition.

In some embodiments, a stabilizing agent is not contained or used in themethods of treatment, methods of administration, and dosage regimesdescribed herein.

Pharmaceutical Compositions and Formulations

The compositions described herein may be used in the preparation of aformulation, such as a pharmaceutical formulation, by combining thenanoparticle composition(s) described with a pharmaceutical acceptablecarrier, excipients, stabilizing agents or other agent, which are knownin the art, for use in the methods of treatment, methods ofadministration, and dosage regimes described herein. In someembodiments, the pharmaceutical composition includes nanoparticlescomprising rapamycin or a derivative thereof and a carrier protein(e.g., albumin). In some embodiments, the pharmaceutical compositionincludes a) nanoparticles comprising rapamycin or a derivative thereofand a carrier protein (e.g., albumin) and b) at least one othertherapeutic agent. In some embodiments, the other therapeutic agentcomprises a chemotherapeutic agent (such as any of the chemotherapeuticagents described herein). In some embodiments, the other therapeuticagent comprises a hormone therapeutic agent.

To increase stability by increasing the negative zeta potential ofnanoparticles, certain negatively charged components may be added. Suchnegatively charged components include, but are not limited to bilesalts, bile acids, glycocholic acid, cholic acid, chenodeoxycholic acid,taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholicacid, litocholic acid, ursodeoxycholic acid, dehydrocholic acid, andothers; phospholipids including lecithin (egg yolk) based phospholipidswhich include the following phosphatidylcholines:palmitoyloleoylphosphatidylcholine,palmitoyllinoleoylphosphatidylcholine,stearoyllinoleoylphosphatidylcholine, stearoyloleoylphosphatidylcholine,stearoylarachidoylphosphatidylcholine, anddipalmitoylphosphatidylcholine. Other phospholipids includingL-α-dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine(DOPC), distearoylphosphatidylcholine (DSPC), hydrogenated soyphosphatidylcholine (HSPC), and other related compounds. Negativelycharged surfactants or emulsifiers are also suitable as additives, e.g.,sodium cholesteryl sulfate and the like.

In some embodiments, the composition is suitable for administration to ahuman. In some embodiments, the composition is suitable foradministration to a mammal such as, in the veterinary context, domesticpets and agricultural animals. There are a wide variety of suitableformulations of the inventive composition (see, e.g., U.S. Pat. Nos.5,916,596 and 6,096,331, which are hereby incorporated by reference intheir entireties). The following formulations and methods are merelyexemplary and are in no way limiting. Formulations suitable for oraladministration can comprise (a) liquid solutions, such as an effectiveamount of the compound dissolved in diluents, such as water, saline, ororange juice, (b) capsules, sachets or tablets, each containing apredetermined amount of the active ingredient, as solids or granules,(c) suspensions in an appropriate liquid, (d) suitable emulsions, and(e) powders. Tablet forms can include one or more of lactose, mannitol,corn starch, potato starch, microcrystalline cellulose, acacia, gelatin,colloidal silicon dioxide, croscarmellose sodium, talc, magnesiumstearate, stearic acid, and other excipients, colorants, diluents,buffering agents, moistening agents, preservatives, flavoring agents,and pharmacologically compatible excipients. Lozenge forms can comprisethe active ingredient in a flavor, usually sucrose and acacia ortragacanth, as well as pastilles comprising the active ingredient in aninert base, such as gelatin and glycerin, or sucrose and acacia,emulsions, gels, and the like containing, in addition to the activeingredient, such excipients as are known in the art.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation compatible with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizing agents, andpreservatives. The formulations can be presented in unit-dose ormulti-dose sealed containers, such as ampules and vials, and can bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid excipient methods of treatment, methodsof administration, and dosage regimes described herein (i.e., water) forinjection, immediately prior to use. Extemporaneous injection solutionsand suspensions can be prepared from sterile powders, granules, andtablets of the kind previously described. Injectable formulations arepreferred.

Formulations suitable for aerosol administration comprise the inventivecomposition include aqueous and non-aqueous, isotonic sterile solutions,which can contain anti-oxidants, buffers, bacteriostats, and solutes, aswell as aqueous and non-aqueous sterile suspensions that can includesuspending agents, solubilizers, thickening agents, stabilizing agents,and preservatives, alone or in combination with other suitablecomponents, which can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They also canbe formulated as pharmaceuticals for non-pressured preparations, such asin a nebulizer or an atomizer.

In some embodiments, the composition is formulated to have a pH in therange of about 4.5 to about 9.0, including for example pH ranges of anyof about 5.0 to about 8.0, about 6.5 to about 7.5, and about 6.5 toabout 7.0. In some embodiments, the pH of the composition is formulatedto no less than about 6, including for example no less than about any of6.5, 7, or 8 (e.g., about 8). The composition can also be made to beisotonic with blood by the addition of a suitable tonicity modifier,such as glycerol.

The nanoparticles of this invention can be enclosed in a hard or softcapsule, can be compressed into tablets, or can be incorporated withbeverages or food or otherwise incorporated into the diet. Capsules canbe formulated by mixing the nanoparticles with an inert pharmaceuticaldiluent and inserting the mixture into a hard gelatin capsule of theappropriate size. If soft capsules are desired, a slurry of thenanoparticles with an acceptable vegetable oil, light petroleum or otherinert oil can be encapsulated by machine into a gelatin capsule.

Also provided are unit dosage forms comprising the compositions andformulations described herein. These unit dosage forms can be stored ina suitable packaging in single or multiple unit dosages and may also befurther sterilized and sealed. For example, the pharmaceuticalcomposition (e.g., a dosage or unit dosage form of a pharmaceuticalcomposition) may include (i) nanoparticles that comprise rapamycin or aderivative thereof and a carrier protein and (ii) a pharmaceuticallyacceptable carrier. In other examples, the pharmaceutical composition(e.g., a dosage or unit dosage form of a pharmaceutical compositionincludes a) nanoparticles comprising rapamycin or a derivative thereofand a carrier protein (e.g., albumin) and b) at least one othertherapeutic agent. In some embodiments, the other therapeutic agentcomprises a chemotherapeutic agent (such as any of the chemotherapeuticagents described herein). In some embodiments, the other therapeuticagent comprises a hormone therapeutic agent. In some embodiments, thepharmaceutical composition also includes one or more other compounds (orpharmaceutically acceptable salts thereof) that are useful for treatingcancer. In various embodiments, the amount of rapamycin or a derivativethereof in the composition is included in any of the following ranges:about 20 to about 50 mg, about 50 to about 100 mg, about 100 to about125 mg, about 125 to about 150 mg, about 150 to about 175 mg, about 175to about 200 mg, about 200 to about 225 mg, about 225 to about 250 mg,about 250 to about 300 mg, or about 300 to about 350 mg. In someembodiments, the amount of rapamycin or derivative thereof in thecomposition (e.g., a dosage or unit dosage form) is in the range ofabout 54 mg to about 540 mg, such as about 180 mg to about 270 mg orabout 216 mg, of the rapamycin or derivative thereof. In someembodiments, the carrier is suitable for parental administration (e.g.,intravenous administration). In some embodiments, a taxane is notcontained in the composition. In some embodiments, the rapamycin orderivative thereof is the only pharmaceutically active agent for thetreatment of cancer that is contained in the composition.

In some embodiments, the invention features a dosage form (e.g., a unitdosage form) for the treatment of cancer comprising (i) nanoparticlesthat comprise a carrier protein and rapamycin or a derivative thereof,wherein the amount of rapamycin or derivative thereof in the unit dosagefrom is in the range of about 180 mg to about 270 mg, and (ii) apharmaceutically acceptable carrier. In some embodiments, the amount ofthe rapamycin or derivative thereof in the unit dosage form includesabout 216 mg.

Also provided are articles of manufacture comprising the compositions,formulations, and unit dosages described herein in suitable packagingfor use in the methods of treatment, methods of administration, anddosage regimes described herein. Suitable packaging for compositionsdescribed herein are known in the art, and include, for example, vials(such as sealed vials), vessels (such as sealed vessels), ampules,bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags),and the like. These articles of manufacture may further be sterilizedand/or sealed.

Kits

The invention also provides kits comprising the compositions,formulations, unit dosages, and articles of manufacture described hereinfor use in the methods of treatment, methods of administration, anddosage regimes described herein. Kits of the invention include one ormore containers comprising rapamycin or a derivative thereof-containingnanoparticle compositions (formulations or unit dosage forms and/orarticles of manufacture), and in some embodiments, further compriseinstructions for use in accordance with any of the methods of treatmentdescribed herein. In some embodiments, the kit further comprises atleast one other therapeutic agent. In some embodiments, the othertherapeutic agent comprises a chemotherapeutic agent (such as any of thechemotherapeutic agents described herein). In some embodiments, theother therapeutic agent comprises a hormone therapeutic agent. In someembodiments, the kit comprises i) a composition comprising nanoparticlescomprising a rapamycin and a carrier protein (such as albumin) and ii)instructions for administering the nanoparticles and thechemotherapeutic agents simultaneously and/or sequentially, fortreatment of cancer. In various embodiments, the cancer is early stagecancer, non-metastatic cancer, primary cancer, advanced cancer, stage 1Vcancer, locally advanced cancer, metastatic cancer, cancer in remission,recurrent cancer, cancer in an adjuvant setting, cancer in a neoadjuvantsetting, or cancer substantially refractory to hormone treatment. Invarious embodiments, the amount of rapamycin or a derivative thereof inthe kit is included in any of the following ranges: about 20 to about 50mg, about 50 to about 100 mg, about 100 to about 125 mg, about 125 toabout 150 mg, about 150 to about 175 mg, about 175 to about 200 mg,about 200 to about 225 mg, about 225 to about 250 mg, about 250 to about300 mg, or about 300 to about 350 mg. In some embodiments, the amount ofrapamycin or a derivative thereof in the kit is in the range of about 54mg to about 540 mg, such as about 180 mg to about 270 mg or about 216mg. In some embodiments, the kit includes one or more other compounds(i.e., one or more compounds other than a taxane) that are useful fortreating cancer. In some embodiments, the other compound is achemotherapeutic agent. In some embodiments, the other compound is ahormone therapeutic.

Instructions supplied in the kits of the invention are typically writteninstructions on a label or package insert (e.g., a paper sheet includedin the kit), but machine-readable instructions (e.g., instructionscarried on a magnetic or optical storage disk) are also acceptable. Theinstructions relating to the use of the nanoparticle compositionsgenerally include information as to dosage, dosing schedule, and routeof administration for the intended treatment. In some embodiments, theinstructions comprise instructions for providing a first and secondtherapy, wherein either the first or second therapy comprisesadministering a composition that comprises nanoparticles of rapamycin orderivative thereof and a carrier protein. The kit may further comprise adescription of selecting an individual suitable or treatment.

The present invention also provides kits comprising compositions (orunit dosages forms and/or articles of manufacture) described herein andmay further comprise instruction(s) on methods of using the composition,such as uses further described herein. In some embodiments, the kit ofthe invention comprises the packaging described above. In otherembodiments, the kit of the invention comprises the packaging describedabove and a second packaging comprising a buffer. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, syringes, andpackage inserts with instructions for performing any methods describedherein.

For combination therapies of the invention, the kit may containinstructions for administering the first and second therapiessimultaneously and/or sequentially for the effective treatment ofcancer. The first and second therapies can be present in separatecontainers or in a single container. It is understood that the kit maycomprise one distinct composition or two or more compositions whereinone composition comprises a first therapy and one composition comprisesa second therapy.

Kits may also be provided that contain sufficient dosages of rapamycinor a derivative thereof as disclosed herein to provide effectivetreatment for an individual for an extended period, such as any of aweek, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5months, 6 months, 7 months, 8 months, 9 months or more. Kits may alsoinclude multiple unit doses of rapamycin or a derivative thereofcompositions, pharmaceutical compositions, and formulations describedherein and instructions for use and packaged in quantities sufficientfor storage and use in pharmacies, for example, hospital pharmacies andcompounding pharmacies. In some embodiments, the kit comprises a dry(e.g., lyophilized) composition that can be reconstituted, resuspended,or rehydrated to form generally a stable aqueous suspension ofnanoparticles comprising rapamycin or a derivative thereof and albumin(e.g., rapamycin or a derivative thereof coated with albumin).

The kits of the invention are in suitable packaging. Suitable packaginginclude, but is not limited to, vials, bottles, jars, flexible packaging(e.g., seled Mylar or plastic bags), and the like. Kits may optionallyprovide additional components such as buffers and interpretativeinformation.

Methods of Making the Compositions

Methods of making compositions containing carrier proteins and poorlywater soluble pharmaceutical agents are known in the art. For example,nanoparticles containing poorly water soluble pharmaceutical agents andcarrier proteins (e.g., albumin) can be prepared under conditions ofhigh shear forces (e.g., sonication, high pressure homogenization, orthe like). These methods are disclosed in, for example, U.S. Pat. Nos.5,916,596; 6,506,405; and 6,537,579 and also in U.S. Pat. Pub. No.2005/0004002A1, which are each hereby incorporated by reference in theirentireties.

Briefly, the rapamycin or derivative hereof is dissolved in an organicsolvent. Suitable organic solvents include, for example, ketones,esters, ethers, chlorinated solvents, and other solvents known in theart. For example, the organic solvent can be methylene chloride,chloroform/ethanol, or chloroform/t-butanol (for example with a ratio ofabout any of 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, or 9:1 or with a ratio of about any of 3:7, 5:7,4:6, 5:5, 6:5, 8:5, 9:5, 9.5:5, 5:3, 7:3, 6:4, or 9.5:0.5). The solutionis added to a carrier protein (e.g., human serum albumin). The mixtureis subjected to high pressure homogenization (e.g., using an Avestin,APV Gaulin, Microfluidizer™ such as a Microfluidizer™ Processor M-110EHfrom Microfluidics, Stansted, or Ultra Turrax homogenizer). The emulsionmay be cycled through the high pressure homogenizer for between about 2to about 100 cycles, such as about 5 to about 50 cycles or about 8 toabout 20 cycles (e.g., about any of 8, 10, 12, 14, 16, 18 or 20 cycles).The organic solvent can then be removed by evaporation utilizingsuitable equipment known for this purpose, including, but not limitedto, rotary evaporators, falling film evaporators, wiped filmevaporators, spray driers, and the like that can be operated in batchmode or in continuous operation. The solvent may be removed at reducedpressure (such as at about any of 25 mm Hg, 30 mm Hg, 40 mm Hg, 50 mmHg, 100 mm Hg, 200 mm Hg, or 300 mm Hg). The amount of time used toremove the solvent under reduced pressure may be adjusted based on thevolume of the formulation. For example, for a formulation produced on a300 mL scale, the solvent can be removed at about 1 to about 300 mm Hg(e.g., about any of 5-100 mm Hg, 10-50 mm Hg, 20-40 mm Hg, or 25 mm Hg)for about 5 to about 60 minutes (e.g., about any of 7, 8, 9, 10, 11, 12,13, 14, 15 16, 18, 20, 25, or 30 minutes).

If desired, human albumin solution may be added to the dispersion toadjust the human serum albumin to rapamycin ratio or to adjust theconcentration of rapamycin in the dispersion. For example, human serumalbumin solution (e.g., 25% w/v) can be added to adjust the human serumalbumin to rapamycin ratio to about any of 18:1, 15:1 14:1, 13:1, 12:1,11:1, 10:1, 9:1, 8:1, 7.5:1, 7:1, 6:1, 5:1, 4:1, or 3:1. For example,human serum albumin solution (e.g., 25% w/v) or another solution isadded to adjust the concentration of rapamycin in the dispersion toabout any of 0.5 mg/ml, 1.3 mg/ml, 1.5 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml,5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 15 mg/ml, 20mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, or 50 mg/ml. The dispersion may beserially filtered through multiple filters, such as a combination of 1.2μm and 0.8/0.2 μm filters; the combination of 1.2 μm, 0.8 μm, 0.45 μm,and 0.22 μm filters; or the combination of any other filters known inthe art. The dispersion obtained can be further lyophilized. Thenanoparticle compositions may be made using a batch process or acontinuous process (e.g., the production of a composition on a largescale).

If desired, a second therapy (e.g., one or more compounds useful fortreating breast cancer), an antimicrobial agent, sugar, and/orstabilizing agent can also be included in the composition. Thisadditional agent can either be admixed with the rapamycin and/or thecarrier protein during preparation of the rapamycin/carrier proteincomposition, or added after the rapamycin/carrier protein composition isprepared. For example, the agent can be added along with an aqueousmedium used to reconstitute/suspend the rapamycin/carrier proteincomposition or added to an aqueous suspension of the carrierprotein-associated rapamycin. In some embodiments, the agent is admixedwith the rapamycin/carrier protein composition prior to lyophilization.In some embodiments, the agent is added to the lyophilizedpharmaceutical agent/carrier protein composition. In some embodimentswhen the addition of the agent changes the pH of the composition, the pHin the composition are generally (but not necessarily) adjusted to adesired pH. Exemplary pH values of the compositions include, forexample, in the range of about 5 to about 8.5. In some embodiments, thepH of the composition is adjusted to no less than about 6, including forexample no less than any of about 6.5, 7, or 8 (e.g., about 8).

The invention also provides methods of making the combination therapiesdescribed herein for use in the treatment of cancer. For example, thereis provided a method of preparing a composition comprising rapamycin ora derivative thereof, a carrier protein (e.g., albumin), and a secondtherapy by combining (e.g., admixing) a composition containing rapamycin(or a derivative thereof) and a carrier protein with a second therapy(e.g., one or more other pharmaceutically active agents for thetreatment of cancer). If desired, an antimicrobial agent, sugar, and/orstabilizing agent can also be included in the composition.

Unless defined otherwise, the meanings of all technical and scientificterms used herein are those commonly understood by one of skill in theart to which this invention belongs. One of skill in the art will alsoappreciate that any methods and materials similar or equivalent to thosedescribed herein can also be used to practice or test the invention.

The specification is most thoroughly understood in light of thereferences cited herein. The disclosures of all publications, patents,patent applications, and published patent applications referred toherein are each hereby incorporated herein by reference in theirentireties.

The following Examples are provided to illustrate, but not limit, theinvention.

EXAMPLES

The examples, which are intended to be purely exemplary of the inventionand should therefore not be considered to limit the invention in anyway, also describe and detail aspects and embodiments of the inventiondiscussed above. The examples are not intended to represent that theexperiments below are all or the only experiments performed. Effortshave been made to ensure accuracy with respect to numbers used (forexample, amounts, temperature, etc.) but some experimental errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, molecular weight is weight average molecularweight, temperature is in degrees Centigrade, and pressure is at or nearatmospheric.

Example 1 Exemplary Methods for the Formation of NanoparticleCompositions with Rapamycin and Albumin Example 1-A

This example demonstrates the preparation of a pharmaceuticalcomposition comprising rapamycin and albumin in which the rapamycinconcentration was 8 mg/mL in the emulsion and the formulation was madeon a 300 mL scale. Rapamycin (2400 mg) was dissolved in 12 mL ofchloroform/t-butanol. The solution was then added into 288 mL of a humanserum albumin solution (3% w/v). The mixture was homogenized for 5minutes at 10,000 rpm (Vitris homogenizer model Tempest I.Q.) in orderto form a crude emulsion, and then transferred into a high pressurehomogenizer. The emulsification was performed at 20,000 psi whilerecycling the emulsion. The resulting system was transferred into aRotavap, and the solvent was rapidly removed at 40° C. at reducedpressure (25 mm of Hg). The resulting dispersion was translucent. Atthis stage, human serum albumin solution was added to the dispersion toadjust the human serum albumin to rapamycin ratio. The dispersion wasserially filtered through multiple filters. The size of the filteredformulation was 85-100 nm (Z_(av), Malvern Zetasizer). The dispersionwas further lyophilized (FTS Systems, Dura-Dry μP, Stone Ridge, NewYork) for 60 hours. The resulting cake was easily reconstitutable to theoriginal dispersion by the addition of sterile water or 0.9% (w/v)sterile saline. The particle size after reconstitution was the same asbefore lyophilization.

Example 1-B

This example demonstrates the preparation of a pharmaceuticalcomposition comprising rapamycin and albumin in which the rapamycinconcentration was 8.3 mg/mL in the emulsion and the formulation was madeon a 200 mL scale. Rapamycin (1660 mg) was dissolved in 8.5 mL ofchloroform/ethanol. The solution was then added into 191.5 mL of a humanserum albumin solution (6% w/v). The mixture was homogenized for 5minutes at 10,000 rpm (Vitris homogenizer model Tempest I.Q.) in orderto form a crude emulsion, and then transferred into a high pressurehomogenizer. The emulsification was performed at 20,000 psi whilerecycling the emulsion. The resulting system was transferred into aRotavap, and the solvent was rapidly removed at 40° C. at reducedpressure (25 mm of Hg). The dispersion was serially filtered. The sizeof the 0.22 μm filtered formulation was 85 nm (Z_(av), MalvernZetasizer). The dispersion was further lyophilized (FTS Systems,Dura-Dry μP, Stone Ridge, New York) for 60 hours. The resulting cake waseasily reconstitutable to the original dispersion by addition of 0.9%(w/v) sterile saline. The particle size after reconstitution was thesame as before lyophilization.

Example 1-C

This example demonstrates the preparation of a pharmaceuticalcomposition comprising rapamycin and albumin in which the rapamycinconcentration was 16.2 mg/mL in the emulsion and the formulation wasmade on a 200 mL scale. Rapamycin (3240 mg) was dissolved in 16 mL ofchloroform/ethanol. The solution was then added into 184 mL of a humanserum albumin solution (6% w/v). The mixture was homogenized for 5minutes at 10,000 rpm (Vitris homogenizer model Tempest I.Q.) in orderto form a crude emulsion, and then transferred into a high pressurehomogenizer. The emulsification was performed at 20,000 psi whilerecycling the emulsion. The resulting system was transferred into aRotavap, and the solvent was rapidly removed at 40° C. at reducedpressure (25 mm of Hg). At this stage, human serum albumin solution wasadded to the dispersion and the volume of the dispersion was made to 400mL to adjust the human serum albumin to rapamycin ratio and to adjustthe rapamycin concentration. The dispersion was serially filtered. Thesize of the 0.22 μm filtered formulation was 99 nm (Z_(av), MalvernZetasizer). The dispersion was further lyophilized (FTS Systems,Dura-Dry μP, Stone Ridge, New York) for 60 hours. The resulting cake waseasily reconstitutable to the original dispersion by addition of 0.9%(w/v) sterile saline. The particle size after reconstitution was thesame as before lyophilization.

Example 1-D

This example demonstrates the preparation of a pharmaceuticalcomposition comprising rapamycin and albumin in which the rapamycinconcentration was 8.2 mg/mL in the emulsion and the formulation was madeon a 40 mL scale. Rapamycin (328 mg) was dissolved in 1.8 mL ofchloroform/ethanol. The solution was then added into 38.2 mL of a humanserum albumin solution (6% w/v). The mixture was homogenized for 5minutes at 10,000 rpm (Vitris homogenizer model Tempest I.Q.) in orderto form a crude emulsion, and then transferred into a high pressurehomogenizer. The emulsification was performed at 20,000 psi whilerecycling the emulsion. The resulting system was transferred into aRotavap, and the solvent was rapidly removed at 40° C. at reducedpressure (40 mm of Hg). The dispersion was serially filtered. The sizeof the 0.22 μm filtered formulation was 108 nm (Z_(av), MalvernZetasizer). The liquid suspension was found to be stable at 4° C. and25° C. at least for 48 hours.

Example 1-E

This example demonstrates the preparation of a pharmaceuticalcomposition comprising rapamycin and albumin in which the rapamycinconcentration was 8.5 mg/mL in the emulsion and the formulation was madeon a 30 mL scale. Rapamycin (255 mg) was dissolved in 1.35 mL ofchloroform/ethanol. The solution was then added into 28.7 mL of a humanserum albumin solution (6% w/v). The mixture was homogenized for 5minutes at 10,000 rpm (Vitris homogenizer model Tempest I.Q.) in orderto form a crude emulsion, and then transferred into a high pressurehomogenizer. The emulsification was performed at 20,000 psi whilerecycling the emulsion. The resulting system was transferred into aRotavap, and the solvent was rapidly removed at 40° C. at reducedpressure (40 mm of Hg). The dispersion was serially filtered. The sizeof the 0.22 μm filtered formulation was 136 nm (Z_(av), MalvernZetasizer). The liquid suspension was found to be stable at 4° C. and25° C. at least for 24 hours.

Example 1-F

This example demonstrates the preparation of a pharmaceuticalcomposition comprising rapamycin and albumin in which the rapamycinconcentration was 9.2 mg/mL in the emulsion and the formulation was madeon a 20 mL scale. Rapamycin (184 mg) was dissolved in 1.0 mL ofchloroform/ethanol. The solution was then added into 19.0 mL of a humanserum albumin solution (7% w/v). The mixture was homogenized for 5minutes at 10,000 rpm (Vitris homogenizer model Tempest I.Q.) in orderto form a crude emulsion, and then transferred into a high pressurehomogenizer. The emulsification was performed at 20,000 psi whilerecycling the emulsion. The resulting system was transferred into aRotavap, and the solvent was rapidly removed at 40° C. at reducedpressure (40 mm of Hg). The dispersion was serially filtered. The sizeof the 0.22 μm filtered formulation was 124 nm (Z_(av), MalvernZetasizer). The liquid suspension was found to be stable at 4° C. and25° C. at least for 24 hours.

Example 1-G

This example demonstrates the preparation of a pharmaceuticalcomposition comprising rapamycin and albumin in which the rapamycinconcentration was 8.4 mg/mL in the emulsion and the formulation was madeon a 20 mL scale. Rapamycin (168 mg) was dissolved in 1.2 mL ofchloroform/ethanol. The solution was then added into 18.8 mL of a humanserum albumin solution (6% w/v). The mixture was homogenized for 5minutes at 10,000 rpm (Vitris homogenizer model Tempest I.Q.) in orderto form a crude emulsion, and then transferred into a high pressurehomogenizer. The emulsification was performed at 20,000 psi whilerecycling the emulsion. The resulting system was transferred into aRotavap, and the solvent was rapidly removed at 40° C. at reducedpressure (40 mm of Hg). The dispersion was serially filtered. The sizeof the 0.22 μm filtered formulation was 95 nm (Z_(av), MalvernZetasizer).

Example 1-H

This example demonstrates the preparation of a pharmaceuticalcomposition comprising rapamycin and albumin in which the rapamycinconcentration was 8.2 mg/mL in the emulsion and the formulation was madeon a 20 mL scale. Rapamycin (164 mg) was dissolved in 0.9 mL ofchloroform/ethanol. The solution was then added into 19.1 mL of a humanserum albumin solution (8% w/v). The mixture was homogenized for 5minutes at 10,000 rpm (Vitris homogenizer model Tempest I.Q.) in orderto form a crude emulsion, and then transferred into a high pressurehomogenizer. The emulsification was performed at 20,000 psi whilerecycling the emulsion. The resulting system was transferred into aRotavap, and the solvent was rapidly removed at 40° C. at reducedpressure (40 mm of Hg). The dispersion was serially filtered. The sizeof the 0.22 μm filtered formulation was 149 nm (Z_(av), MalvernZetasizer).

Example 1-I

This example demonstrates the preparation of a pharmaceuticalcomposition comprising rapamycin and albumin in which the rapamycinconcentration was 6.6 mg/mL in the emulsion and the formulation was madeon a 20 mL scale. Rapamycin (132 mg) was dissolved in 0.8 mL ofchloroform/ethanol. The solution was then added into 19.2 mL of a humanserum albumin solution (5% w/v). The mixture was homogenized for 5minutes at 10,000 rpm (Vitris homogenizer model Tempest I.Q.) in orderto form a crude emulsion, and then transferred into a high pressurehomogenizer. The emulsification was performed at 20,000 psi whilerecycling the emulsion. The resulting system was transferred into aRotavap, and the solvent was rapidly removed at 40° C. at reducedpressure (40 mm of Hg). The dispersion was serially filtered. The sizeof the 0.22 μm filtered formulation was 129 nm (Z_(av), MalvernZetasizer).

Example 1-J

This example demonstrates the preparation of a pharmaceuticalcomposition comprising rapamycin and albumin in which the rapamycinconcentration was 4.0 mg/mL in the emulsion and the formulation was madeon a 20 mL scale. Rapamycin (80 mg) was dissolved in 0.8 mL ofchloroform/ethanol. The solution was then added into 19.2 mL of a humanserum albumin solution (3% w/v). The mixture was homogenized for 5minutes at 10,000 rpm (Vitris homogenizer model Tempest I.Q.) in orderto form a crude emulsion, and then transferred into a high pressurehomogenizer. The emulsification was performed at 20,000 psi whilerecycling the emulsion. The resulting system was transferred into aRotavap, and the solvent was rapidly removed at 40° C. at reducedpressure (40 mm of Hg). The dispersion was serially filtered. The sizeof the 0.22 μm filtered formulation was 108 nm (Z_(av), MalvernZetasizer).

Example 1-K

This example demonstrates the preparation of a pharmaceuticalcomposition comprising rapamycin and albumin in which the rapamycinconcentration was 4.0 mg/mL in the emulsion and the formulation was madeon a 20 mL scale. Rapamycin (80 mg) was dissolved in 0.8 mL ofchloroform/ethanol. The solution was then added into 19.2 mL of a humanserum albumin solution (1% w/v). The mixture was homogenized for 5minutes at 10,000 rpm (Vitris homogenizer model Tempest I.Q.) in orderto form a crude emulsion, and then transferred into a high pressurehomogenizer. The emulsification was performed at 20,000 psi whilerecycling the emulsion. The resulting system was transferred into aRotavap, and the solvent was rapidly removed at 40° C. at reducedpressure (40 mm of Hg). The dispersion was serially filtered. The sizeof the 0.22 μm filtered formulation was 99 nm (Z_(av), MalvernZetasizer).

Example 1-L

This example demonstrates the preparation of a pharmaceuticalcomposition comprising rapamycin and albumin in which the rapamycinconcentration was 5.0 mg/mL in the emulsion and the formulation was madeon a 20 mL scale. Rapamycin (100 mg) was dissolved in 0.8 mL ofchloroform/ethanol. The solution was then added into 19.2 mL of a humanserum albumin solution (3% w/v). The mixture was homogenized for 5minutes at 10,000 rpm (Vitris homogenizer model Tempest I.Q.) in orderto form a crude emulsion, and then transferred into a high pressurehomogenizer. The emulsification was performed at 20,000 psi whilerecycling the emulsion. The resulting system was transferred into aRotavap, and the solvent was rapidly removed at 40° C. at reducedpressure (40 mm of Hg). The dispersion was serially filtered. The sizeof the 0.22 μm filtered formulation was 146 nm (Z_(av), MalvernZetasizer).

Example 1-M

This example demonstrates the preparation of a pharmaceuticalcomposition comprising rapamycin and albumin in which the rapamycinconcentration was 4.0 mg/mL in the emulsion and the formulation was madeon a 20 mL scale. Rapamycin (80 mg) was dissolved in 0.8 mL ofchloroform/ethanol. The solution was then added into 19.2 mL of a humanserum albumin solution (3% w/v). The mixture was homogenized for 5minutes at 10,000 rpm (Vitris homogenizer model Tempest I.Q.) in orderto form a crude emulsion, and then transferred into a high pressurehomogenizer. The emulsification was performed at 20,000 psi whilerecycling the emulsion. The resulting system was transferred into aRotavap, and the solvent was rapidly removed at 40° C. at reducedpressure (40 mm of Hg). The resulting dispersion was a white milkysuspension. The dispersion was serially filtered. The size of the 0.22μm filtered formulation was 129 nm (Z_(av), Malvern Zetasizer).

Example 1-N

This example demonstrates the preparation of a pharmaceuticalcomposition comprising rapamycin and albumin in which the rapamycinconcentration was 4.0 mg/mL in the emulsion and the formulation was madeon a 20 mL scale. Rapamycin (80 mg) was dissolved in 0.8 mL ofchloroform/ethanol. The solution was then added into 19.2 mL of a humanserum albumin solution (3% w/v). The mixture was homogenized for 5minutes at 10,000 rpm (Vitris homogenizer model Tempest I.Q.) in orderto form a crude emulsion, and then transferred into a high pressurehomogenizer. The emulsification was performed at 20,000 psi whilerecycling the emulsion. The resulting system was transferred into aRotavap, and the solvent was rapidly removed at 40° C. at reducedpressure (40 mm of Hg). The dispersion was serially filtered. The sizeof the 0.22 μm filtered formulation was 166 nm (Z_(av), MalvernZetasizer).

Example 1-O

This example demonstrates the preparation of a pharmaceuticalcomposition comprising rapamycin and albumin in which the rapamycinconcentration was 4.0 mg/mL in the emulsion and the formulation was madeon a 20 mL scale. Rapamycin (80 mg) was dissolved in 0.8 mL ofchloroform/ethanol. The solution was then added into 19.2 mL of a humanserum albumin solution (3% w/v). The mixture was homogenized for 5minutes at 10,000 rpm (Vitris homogenizer model Tempest I.Q.) in orderto form a crude emulsion, and then transferred into a high pressurehomogenizer. The emulsification was performed at 20,000 psi whilerecycling the emulsion. The resulting system was transferred into aRotavap, and the solvent was rapidly removed at 40° C. at reducedpressure (40 mm of Hg). The dispersion was serially filtered. The sizeof the 0.22 μm filtered formulation was 90 nm (Z_(av), MalvernZetasizer).

Example 1-P

This example demonstrates the preparation of a pharmaceuticalcomposition comprising rapamycin and albumin in which the rapamycinconcentration was 4.0 mg/mL in the emulsion and the formulation was madeon a 20 mL scale. Rapamycin (80 mg) was dissolved in 0.8 mL ofchloroform/ethanol. The solution was then added into 19.2 mL of a humanserum albumin solution (3% w/v). The mixture was homogenized for 5minutes at 10,000 rpm (Vitris homogenizer model Tempest I.Q.) in orderto form a crude emulsion, and then transferred into a high pressurehomogenizer. The emulsification was performed at 20,000 psi whilerecycling the emulsion. The resulting system was transferred into aRotavap, and the solvent was rapidly removed at 40° C. at reducedpressure (40 mm of Hg). The dispersion was serially filtered. The sizeof the 0.22 μm filtered formulation was 81 nm (Z_(av), MalvernZetasizer).

Example 1-Q

This example demonstrates the preparation of a pharmaceuticalcomposition comprising rapamycin and albumin. Rapamycin (30 mg) wasdissolved in 2 ml chloroform/ethanol. The solution was then added into27.0 ml of a human serum albumin solution (3% w/v). The mixture washomogenized for 5 minutes at low RPM (Vitris homogenizer model TempestI.Q.) in order to form a crude emulsion, and then transferred into ahigh pressure homogenizer. The emulsification was performed at9000-40,000 psi while recycling the emulsion for at least 5 cycles. Theresulting system was transferred into a Rotavap, and the solvent wasrapidly removed at 40° C. at reduced pressure (30 mm Hg) for 20-30minutes. The resulting dispersion was translucent, and the typicalaverage diameter of the resulting particles was in the range 50-220 nm(Z-average, Malvern Zetasizer). The dispersion was further lyophilizedfor 48 hours. The resulting cake was easily reconstituted to theoriginal dispersion by addition of sterile water or saline. The particlesize after reconstitution was the same as before lyophilization.

If desired, other compositions of the invention (e.g., compositions thatcontain rapamycin derivatives or carrier proteins other than human serumalbumin) can be made using these methods or a variation of thesemethods. It should be recognized that the amounts, types, andproportions of drug, solvents, and proteins used in these examples arenot limiting in any way.

Example 2A Toxicology and Pharmacokinetic Studies of Nab-rapamycin

The overall toxicity of Nab-rapamycin was determined in a dose rangingstudy in Sprague Dawley rats. The dose levels of Nab-rapamycin used were0, 15, 30, 45, 90 and 180 mg/kg with a q4dx3 schedule. Thepharmacokinetics of Nab-rapamycin was also investigated in SpragueDawley rats at dose levels of 1 (N=3), 15 (N=4), 30 (N=3), and 45 mg/kg(N=4). Blood samples were collected prior to dosing (baseline) andpost-dosing at the following time points: 1, 5, 10, 15, 30 and 45minutes, and 1, 4, 8, 24, 36 and 48 hours. Plasma samples were analyzedfor rapamycin using LC/MS.

Nab-rapamycin was nontoxic at the highest dose of 180 mg/kg on a q4dx3schedule. No changes in blood chemistry or CBC were observed. Nohypercholesterolemia and hypertriglyceridemia were observed. Asillustrated in FIGS. 1 and 2C, Nab-rapamycin exhibited linearpharmacokinetics with respect to dose and rapid extravasculardistribution as demonstrated by large Vss and Vz. The Cmax and AUCinf ofNab-rapamycin were dose proportional (FIGS. 2A and 2B, respectively).

If desired, other compositions of the invention (e.g., compositions thatcontain rapamycin derivatives or carrier proteins other than human serumalbumin) can be tested in these assays for toxicity andpharmacokinetics.

Example 2B Toxicology and Pharmacokinetic Studies of Nab-rapamycin

The overall toxicity of Nab-rapamycin was determined in a dose rangingstudy in Sprague Dawley rats. Nab-rapamycin was intravenouslyadministered at 0, 20, 40, 90, 120, and 180 mg/kg on a q4dx3 schedule ondays 1, 5, and 9 (n=20). Nab-rapamycin was well tolerated at dose levelsup to 90 mg/kg (540 mg/m²) on a q4dx3 schedule. There was 20% and 100%mortality among the highest doses of 120 mg/kg and 180 mg/kg. Nohypercholesterolemia and hypertriglyceridemia were observed.

The pharmacokinetics of Nab-rapamycin was also investigated in SpragueDawley rats at dose levels of 1 (N=5), 15 (N=4), 30 (N=3), and 45 mg/kg(N=4). Blood samples were collected prior to dosing (baseline) andpost-dosing at the following time points: 1, 5, 10, 15, 30 and 45minutes, and 1, 4, 8, 24, 36 and 48 hours. Plasma samples were analyzedfor rapamycin using LC/MS.

Nab-rapamycin exhibited a very rapid distribution phase and large V_(z)and V_(ss). The C_(max) and AUC_(inf) of Nab-rapamycin were doseproportional. See FIG. 1. The PK of Nab-rapamyin is similar toNab-paclitaxel and Nab-docetaxel. FIG. 2D shows the log-linerar plotNab-rapamycin blood concentration vs. time following IV administrationto rats at dose levels of 15 mg/kg, 30 mg/kg, and 45 mg/kg.

Example 3 Inhibition of Breast Cancer Cells Using Nab-rapamycin

The antitumor activity of Nab-rapamycin was examined using a humanmammary carcinoma xenograft in mice. MX-1 tumors were implantedsubcutaneously into both the right and left flanks of female athymicmice (4-5 per group) and allowed to grow to 100 mm³. The mice were thenintravenously administered either saline or Nab-rapamycin at a doselevel of 40 mg/kg with a three times weekly schedule for 4 weeks. Thedosing volume was 2 ml/kg. Tumor growth data were analyzed by ANOVA.

Nab-rapamycin was highly effective against breast cancer, achieving atumor growth inhibition of 88% against the MX-1 xenograft (p<0.0001versus control, ANOVA; FIG. 3A). No significant weight loss was observedin the mice from Nab-rapamycin at 40 mg/kg (FIG. 3B). Thus,Nab-rapamycin was well tolerated even at the highest dose of 180 mg/kgwith a q4dx3 schedule, showed linear pharmacokinetics, and was highlyeffective against a breast cancer model in vivo.

If desired, other compositions of the invention (e.g., compositions thatcontain rapamycin derivatives or carrier proteins other than human serumalbumin) can be tested in this animal model to determine their abilityto treat breast cancer in vivo.

Example 4 Use of Human Clinical Trials to Determine the Ability ofCompositions of the Invention to Treat, Stabilize, Prevent, and/or DelayCancer

If desired, any of the compositions described herein can also be testedin humans to determine the ability of the compositions to treat,stabilize, prevent and/or delay cancer (e.g., breast cancer). Standardmethods can be used for these clinical trials.

In one exemplary method, subjects (e.g., healthy subjects, subjects withcancer such as breast cancer, or subjects at increased risk for cancersuch as breast cancer) are enrolled in a tolerability, pharmacokinetics,and pharmacodynamics phase I study of Nab-rapamycin or a derivativethereof using standard protocols. For example, escalating doses ofrapamycin or a derivative thereof up to about 250 mg/m² as part of acomposition of the invention can be tested. Then a phase II,double-blind randomized controlled trial is performed to determine theefficacy of the Nab-rapamycin or a derivative thereof. If desired, theactivity of Nab-rapamycin or a derivative thereof can be compared tothat of another treatment for cancer (e.g., breast cancer).Alternatively or additionally, the efficacy of a combination ofNab-rapamycin or a derivative thereof and another treatment for cancer(e.g., breast cancer) can be compared to that of either treatment alone.

Example 5 Multiple Myeloma (MM) Cell Lines for Use in Determination ofNab-rapamycin Activity

Interleukin-6 (IL-6) and insulin like growth factor-1 (IGF-1) play a keyrole in the growth, survival, and drug resistance in multiple myeloma(MM) cells. Furthermore, their secretion in bone marrow stromal cells(BMSCs) is up-regulated by adherence of MM cells. IL-6 and IGF-1 mediategrowth of MM cells via activation of the mitogen-activated proteinkinase (MAPK) and phosphatidylinositol 3′-kinase/Akt kinase (PI3-K/Akt)signaling cascades. Several studies show that PI3-K/Akt signalingmediates growth, survival, migration and cell cycle regulation in MM.Activated Akt in turn phosphorylates downstream target molecules,including forkhead transcription factor (FKHR), glycogen synthase kinase(GSK)-3(3, and mammalian target of rapamycin (mTOR).

MM cell lines can be used in standard cell-based assays to test theability of any of the nanoparticle compositions of the invention (e.g.,nanoparticles comprising rapamycin and a carrier protein such asalbumin) to treat MM. The nanoparticle compositions of the invention aredesirable because they may allow rapamycin to be delivered at higherdoses with improved efficacy.

For these cell-based assays, RPMI 8226 and U266 human MM cell lines areobtained from the American Type Culture Collection (ATCC) of Rockville,Md. Patient derived MM cells are purified from patient BM samples, asdescribed by Y. T. Tai, G. Teoh, Y. Shima, et al., J. Immunol. Methods235:11, 2000. Human MM cell lines are cultured in RPMI-1640 media (SigmaChemical, St. Louis, Mo.), containing 10% fetal bovine serum (FBS), 2mmol/L L-glutamine (L-glut, GIBCO, Grand Island, N.Y.), 100 U/mLpenicillin and 100 mg/mL streptomycin (P/S, GIBCO). MM patient cells are95% CD38+, CD45RA−. Bone marrow stromal cells (BMSCs) are prepared fromaspirates of MM patients as well as healthy donors as described by D.Gupta, S. Treon, Y. Shima, et al. in Leukemia, 2001 and S. Gartner andH. S. Kaplan in Proc. Nag. Acad. Sci. USA 77:4756, 1980. Cells arecultured in ISCOVE's modified Dulbecco media containing 20% FBS, 2mmol/L L-glut, and 100 μg/mL P/S. Human umbilical vein endothelial cells(HUVEC P168) are purchased from Clonetics, Biowhittaker, and maintainedin EGM-2MV media (Clonetics, Biowhittaker). The nanoparticles comprisingrapamycin and a carrier protein (such as albumin) are diluted in culturemedium to concentrations ranging, e.g., from 0.01 to 100 μM.

Example 6 Panel of Drug-Resistant MM Cell Lines and Primary MM TumorCells for Use in Determination of Nab-rapamycin Activity

Effectiveness of the nanoparticle compositions of the invention mayfurther be evaluated in drug resistant cell lines. The use of drugresistant cells facilitates the determination of potential cancerpatient subpopulations that may be effectively treated by the use of thenanoparticle compositions of the invention. The activity of any of thenanoparticle compositions of the invention (e.g., nanoparticlescomprising rapamycin and a carrier protein such as albumin) can beevaluated in a panel of drug-sensitive and drug-resistant human MM celllines using standard methods. Exemplary cell lines include adexamethasone (Dex)-sensitive MM-1S cell line, a Dex-resistant MM-1Rcell line; the chemo-sensitive parental MM cell line RPMI-8226/S, andits chemo-resistant sublines RPMI-8226/Dox40 (doxorubicin-resistant),RPMI-8226/MR20 (mitoxantrone-resistant), and RPMI-8226/LR5(melphalan-resistant) cells; MM-1S-TR15 is a TRAIL/Apo2L-resistantsubline; MM-SAR-1 (also referred to as MM-SA-1) cells that are primaryMM tumor cells from a patient resistant to the proteasome inhibitorbortezomib (PS-341) (cells maintained in vitro resistance toPS-341);OCI-My-5 cells; S6B45 cells; ARD; ARK; ARP-1; OPM-1; OPM-6;K620; LP-1; U266; and NCI-H929 cells. All cells are cultured in RPMI1640 medium (Life Technologies, Grand Island, N.Y.) supplemented with10% fetal bovine serum, L-glutamine, penicillin, and streptomycin (LifeTechnologies).

Primary MM tumor cells additionally may be isolated from bone marrow(BM) aspirates of patients, who are resistant to conventional (steroid-and cytotoxic chemotherapy-based) and more recently developed anti-MMagents (e.g. thalidomide or proteasome inhibitors). The resistantprimary MM tumor cells are collected from patients as described above inExample 4.

Example 7 Co-Culture Assays of MM Cells with Bone Marrow Stromal Cells(BMSCs) Treated with Nab-rapamycin

When adhering to BMSCs, MM cells have reduced sensitivity toconventional anti-MM therapies, such as dexamethasone or cytotoxicchemotherapeutics (Chauhan D. et al., Blood 1996, 87, 1104-1112). Thisform of drug resistance is considered a key reason why MM patientseventually relapse when they receive treatment based on administrationof glucocorticoids and/or cytotoxic chemotherapy. Therefore, any of thenanoparticle compositions of the invention (e.g., nanoparticlescomprising rapamycin and a carrier protein such as albumin) can betested to determine whether they overcome the molecular sequelae of theinteraction of BMSCs with MM cells and achieve anti-MM activity in thiscontext. In particular, an in vitro co-culture assay is performed usingMM cells with BMSCs as previously described. BMSCs are grown on 24-wellplates to confluency. Following washings with serum-free medium, primarytumor cells (greater than about 95% purity in CD138+ cells) isolatedfrom MM patients are added to BMSC-coated or control wells as describedpreviously (Uchiyama H. et al., Blood 1993, 82, 3712-3720; Mitsiades N.et al., Blood 2003, 101, 4055-4062) and incubated for 48 hours in thepresence or absence of a nanoparticle composition of the invention, suchas nab-rapamycin. Flow cytometric analysis is performed to detect theCD138+ population of viable MM cells and the effect of the nanoparticlecomposition on MM cell viability is expressed as a percent of viablecells in comparison to the respective vehicle-treated cultures.

Example 8 MTT calorimetric Survival Assay of MM Tissue Culture CellsTreated with Nab-rapamycin

In this example, the effect of nanoparticle composition of the invention(e.g., nanoparticles comprising rapamycin and a carrier protein such asalbumin) on cell viability and survival is assessed. Cell survival isexamined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT; Sigma Chemical, St Louis, Mo.) colorimetric assay, aspreviously described (Mitsiades C. S. et al., Blood 2001, 98, 795-804;Mitsiades N. et al., PNAS 2002, 99, 14374-14379; Mitsiades N. et al.,Blood 2003, 101, 2377-2380). Briefly, cells are plated in 48-well platesat 70% to 80% confluence in the presence of 2.5% fetal bovine serum(FBS) and in the presence of a nanoparticle composition of the invention(e.g., nanoparticles comprising rapamycin and a carrier protein such asalbumin) at final concentration of 0-100 nM rapamycin or DMSO vehiclecontrol. At the end of each treatment, cells are incubated with 1 mg/mLMTT for 4 hours at 37° C. A mixture of isopropanol and 1 N HCl (23:2,vol/vol) is then added under vigorous pipetting to dissolve the formazancrystals. Dye absorbance (A) in viable cells is measured at 570 nm, with630 nm as a reference wavelength. Cell viability is estimated as apercentage of the value of untreated controls. Experiments are typicallyrepeated at least 3 times, and each experimental condition is typicallyrepeated at least in triplicate wells in each experiment. Data isreported are average values +/−SD of representative experiments.

Example 9 Proliferation of MM Cells Treated with Nab-rapamycin

In this example, the effect of nanoparticle composition of the invention(e.g., nanoparticles comprising rapamycin and a carrier protein such asalbumin) on cell proliferation and viability is assessed. Forproliferation and cell viability assays, MM cells are first starved for12 hours in RPMI-1640 media containing 10% fetal bovine serum, and thenplated into 96-well microtiter plates (Costar, Cambridge, Mass.), in thepresence of a nanoparticle composition of the invention (e.g.,nanoparticles comprising rapamycin and a carrier protein such asalbumin) or DMSO control. Proliferation is measured by the incorporationof ³H-thymidine (NEN Products, Boston, Mass.). Specifically, cells arepulsed with ³H-thymidine (0.5.muck/well) for the last 6 hours of 48 hourcultures, harvested onto glass filters with an automatic cell harvester(Cambridge Technology, Cambridge, Mass.), and counted using a LKBBetaplate scintillation counter (Wallac, Gaithersburg, Md.). Measurementof cell viability is performed colorimetrically using a MTS assay,utilizing the CellTiter96 One Solution Reagent (Promega, Madison, Wis.).Cells are exposed to the MTS for the last 2 hours of 48 hour cultures,and absorbance is measured using an ELISA plate reader (MolecularDevices Corp., Sunnyvale, Calif.) at OD of 570 nm.

Example 10 Cell Cycle Analysis of MM Tissue Culture Cells Treated withNab-rapamycin

In this example, the effect of nanoparticle composition of the invention(e.g., nanoparticles comprising rapamycin and a carrier protein such asalbumin) on cell cycle is assessed. MM cells (1×10⁶ cells) are culturedin the presence of a nanoparticle composition of the invention (e.g.,nanoparticles comprising rapamycin and a carrier protein such asalbumin) or DMSO control for 24, 48 and 72 hours. Cells are then washedwith phosphate buffered saline (PBS), fixed with 70% ethanol, andtreated with RNAse (Sigma). Cells are next stained with propidium iodide(PI, 5 μg/mL), and the cell cycle profile is determined using the Msoftware on an Epics flow cytometer (Coulter Immunology, Hialeah, Fla.).

Example 11 Other MM Cell Activity Assays for Cells Treated withNab-rapamycin

Nanoparticle composition of the invention (e.g., nanoparticlescomprising rapamycin and a carrier protein such as albumin) can befurther assessed by other activity assays known in the art. For example,the molecular mechanisms of anti-MM activities of nanoparticlecomposition of the invention may be assessed using, but not limited to,cell cycle profiling by caspases/PARP cleavage and quantification ofanti-apoptotic proteins by Western blotting.

Example 12A Effect of Nab-rapamycin on Human MM Cells in vivo

In this example, the effect of nanoparticle composition of the invention(e.g., nanoparticles comprising rapamycin and a carrier protein such asalbumin) on MM cell growth in vivo is assessed. Mice are inoculatedsubcutaneously into the right flank with 3×107 MM cells in 100 mL ofRPMI 1640, together with 100 μL matrigel basement membrane matrix(Becton Dickinson, Bedford, Mass.). On day 6 post injection, mice areassigned into two treatment groups receiving a nanoparticle compositionof the invention (e.g., nanoparticles comprising rapamycin and a carrierprotein such as albumin) or into a control group. Treatment with ananoparticle composition of the invention is then intravenouslyadministered either saline or nanoparticles comprising rapamycin and acarrier protein such as albumin at a dose level of 40 mg/kg with a threetimes weekly schedule for 4 weeks. The dosing volume is 2 ml/kg. Calipermeasurements of the longest perpendicular tumor diameters are performedtwice per week to estimate the tumor volume. Animals are sacrificed whentheir tumor reached 2 cm or when the mice become moribund. Survival isevaluated from the first day of tumor injection until death.

Example 12B Effect of Nab-Rapamycin on Human MM1S Cells In Vivo

In this example, the effect of Nab-rapamycin on MM1S cell growth in vivowas assessed. Mice were inoculated subcutaneously into the right flankwith 3×10⁷ MM1S cells in 100 mL of RPMI 1640, together with 100 μLmatrigel basement membrane matrix (Becton Dickinson, Bedford, Mass.). Onday 6 post injection, mice were assigned into three treatment groupsreceiving Nab-rapamycin or into a control group. Animals in the controlgroup were administered with 0.9% NaCl solution (i.v.). Animals in thethree treatment groups were administered with Nab-rapamycin at a doseschedule of 20 or 40 mg/kg three times weekly or at a dose schedule of30 mg/kg daily for 15 days. The dosing volume was 2 ml/kg. Calipermeasurements of the longest perpendicular tumor diameters were performedtwice per week to estimate the tumor volume. Animals were sacrificedwhen their tumor reached 2 cm or when the mice became moribund. As shownin FIG. 7, in all three treatment groups, Nab-rapamycin was highlyeffective against multiple myeloma.

Example 13 Cytotoxic Activity of Nab-Rapamycin in Combination withAbraxane™ Against HT29 (Human Colon Carcinoma) Tumor Xenograft

The following example is disclosed in U.S. Ser. No. 11/359,286, whichwas filed Feb. 21, 2006 (i.e., U.S. Pat. Pub. No. 2006/0263434,published Nov. 23, 2006). Nude mice were implanted with 10⁶ HT29 cellson their right flanks. Treatment was initiated once the tumors werepalpable and were greater than 100-200 mm³. The mice were randomlysorted into 4 groups (n=8 per group). Group 1 received saline 3 timesweekly for 4 weeks, i.v.; Group 2 received Abraxane™ at 10 mg/kg, dailyfor 5 days, i.p.; Group 3 received Nab-rapamycin at 40 mg/kg, 3 timesweekly for 4 weeks, i.v.; and Group 4 received both Nab-rapamycin (40mg/kg, 3 times weekly for 4 weeks, i.v.) and Abraxane™ (10 mg/kg, dailyfor 5 days, i.p.). As shoWn in FIG. 4, the tumor suppression was greaterfor the Abraxane™ plus Nab-rapamycin combination therapy than for eithersingle therapy group.

Example 14 Cytotoxic Activity of Nab-Rapamycin Against HT29 (Human ColonCarcinoma) Tumor Xenograft

The antitumor activity of Nab-rapamycin was examined using HT29 humancolon carcinoma xenograft in mice. Male athymic mice (3 per group) wereimplanted with 10⁶ HT29 cells on their right flanks and allowed to growto ˜100 mm³. The mice were then intravenously administered with eitherDMSO at 2 mL/kg or Nab-rapamycin at a dose level of 40 mg/kg with athree times weekly schedule for 4 weeks at a dosing volume of 5 mL/kg.Tumor growth data were analyzed by ANOVA.

Nab-rapamycin significantly inhibited in vivo tumor growth for HT29tumors, achieving a tumor growth inhibition of 78.9% against the HT29tumor xenograft (p=0.005 versus control, ANOVA; FIG. 5A). A −9.2% weightloss was observed in the mice from Nab-rapamycin at 40 mg/kg (FIG. 5B).

Example 15 Cytotoxic Activity of Nab-Rapamacin Against HCT-116 (HumanColon Carcinoma) Tumor Xenograft

The antitumor activity of Nab-rapamycin was examined using HCT-116 humancolon carcinoma xenograft in mice. HCT-116 tumors were implantedsubcutaneously into the right flanks of male athymic nude mice (10 pergroup) and allowed to grow to 100-221 mm³. The mice were thenintravenously administered with either saline or Nab-rapamycin at a doselevel of 40 mg/kg with a three times weekly schedule for 4 weeks at adosing volume of 10 mL/kg. Tumor growth data were analyzed by ANOVA.

Nab-rapamycin significantly inhibited in vivo tumor growth for HCT-116tumors, achieving a tumor growth inhibition of 71% against the HCT-116tumor xenograft (p<0.0001 versus control, ANOVA; FIG. 6A). A −9.7%weight loss was observed in the mice from Nab-rapamycin at 40 mg/kg,which is similar to the −10.7% weight loss for the control group (FIG.6B).

1. A method of treating cancer in an individual, comprisingadministering to the individual an effective amount of a compositioncomprising nanoparticles that comprise rapamycin or a derivative thereofand a carrier protein. 2-3. (canceled)
 4. The method of claim 1, whereinthe cancer is a plasmacytoma.
 5. The method of claim 1, wherein thecancer is selected from the group consisting of multiple myeloma,pancreatic cancer, renal cell carcinoma, prostate cancer, lung cancer,melanoma, and breast cancer.
 6. The method of claim 1, wherein thecancer is not colon cancer.
 7. (canceled)
 8. The method of claim 5,wherein the cancer is pancreatic cancer.
 9. The method of claim 1,wherein the amount of the rapamycin or derivative thereof in theeffective amount of the composition is in the range of about 180 mg toabout 270 mg. 10-15. (canceled)
 16. The method of claim 1, wherein thecarrier protein is albumin.
 17. The method of claim 16, wherein thealbumin is human serum albumin.
 18. The method of claim 1, wherein theaverage diameter of the nanoparticles in the composition is no greaterthan about 200 nm.
 19. (canceled)
 20. The method of claim 1, wherein theindividual is human.
 21. A unit dosage form for treatment of cancercomprising (a) nanoparticles that comprise a carrier protein andrapamycin or a derivative thereof, wherein the amount of the rapamycinor derivative thereof in the unit dosage form is in the range of about180 mg to about 270 mg, and (b) a pharmaceutically acceptable carrier.22-31. (canceled)
 32. A kit comprising (a) nanoparticles that comprise acarrier protein and rapamycin or a derivative thereof, wherein theamount of the rapamycin or derivative thereof in the kit is in the rangeof about 180 mg to about 270 mg, and (b) instructions for using the kitin treating cancer. 33-43. (canceled)
 44. A method of treating cancer inan individual comprising (a) a first therapy comprising administering tothe individual an effective amount of a composition comprisingnanoparticles that comprise rapamycin or a derivative thereof and acarrier protein and (b) a second therapy selected from the groupconsisting of chemotherapy, radiation therapy, surgery, hormone therapy,gene therapy, immunotherapy, bone marrow transplantation, stem celltransplantation, targeted therapy, cryotherapy, ultrasound therapy, andimmunotherapy.
 45. The method of claim 44, wherein the second therapy ischemotherapy.
 46. The method of claim 45, wherein chemotherapy comprisesadministration of a chemotherapeutic selected from the group consistingof antimetabolite agents, platinum-based agents, alkylating agents,tyrosine kinase inhibitors, anthracycline antibiotics, vinca alkloids,proteasome inhibitors, and topoisomerase inhibitors. 47-53. (canceled)54. The method of claim 44, wherein the cancer is selected from thegroup consisting of multiple myeloma, pancreatic cancer, renal cellcarcinoma, prostate cancer, lung cancer, melanoma, and breast cancer.55. The method of claim 44, wherein the cancer is not colon cancer.56-57. (canceled)
 58. The method of claim 44, wherein the amount of therapamycin or derivative thereof in the effective amount of thecomposition is in the range of about 180 mg to about 270 mg. 59-61.(canceled)
 62. The method of claim 44, wherein a taxane is notadministered to the individual. 63-64. (canceled)
 65. The method ofclaim 44, wherein the carrier protein is albumin.
 66. The method ofclaim 44, wherein the albumin is human serum albumin.
 67. The method ofclaim 44, wherein the average diameter of the nanoparticles in thecomposition is no greater than about 200 nm.
 68. (canceled)
 69. Themethod of claim 44, wherein the individual is human.
 70. The method ofclaim 45, wherein the chemotherapy comprises administering to theindividual an inhibitor of the mTOR signaling pathway.
 71. The method ofclaim 45, wherein the chemotherapy comprises administering to theindividual an AKT kinase inhibitor.
 72. The method of claim 72, whereinAKT kinase inhibitor is perifosine.
 73. The method of claim 45, whereinthe chemotherapy comprises administering to the individual a tyrosinekinase inhibitor.
 74. The method of claim 73, wherein the tyrosinekinase inhibitor is erlotinib.